Active-source-pixel, integrated device for rapid analysis of biological and chemical specimens

ABSTRACT

An active-source-pixel, integrated device capable of performing biomolecule detection and/or analysis, such as single-molecule nucleic acid sequencing, is described. An active pixel of the integrated device includes a sample well into which a sample to be analyzed may diffuse, an excitation source for providing excitation energy to the sample well, and a sensor configured to detect emission from the sample. The sensor may comprise two or more segments that produce a set of signals that are analyzed to differentiate between and identify tags that are attached to, or associated with, the sample. Tag differentiation may be spectral and/or temporal based. Identification of the tags may be used to detect, analyze, and/or sequence the biomolecule.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/543,888, filed Nov. 17, 2014, entitled “ACTIVE-SOURCE-PIXEL,INTEGRATED DEVICE FOR RAPID ANALYSIS OF BIOLOGICAL AND CHEMICALSPECIMENS,” which claims priority to U.S. provisional application No.61/905,282, filed Nov. 17, 2013, entitled “INTEGRATED DEVICE FORPROBING, DETECTING AND ANALYZING MOLECULES” and to U.S. provisionalapplication No. 61/917,926, filed Dec. 18, 2013, entitled “INTEGRATEDDEVICE FOR PROBING, DETECTING AND ANALYZING MOLECULES” and to U.S.provisional application No. 61/941,916, filed Feb. 19, 2014, entitled“INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZING MOLECULES.” Theentire disclosures of the foregoing applications are incorporated hereinby reference in their entirety.

FIELD

The present application is directed to devices and methods for analysesof biological and chemical specimens and reactions involving biologicaland chemical samples.

BACKGROUND

Analyses of biological and chemical specimens may be performedconventionally using large, expensive laboratory equipment requiringskilled scientists trained to operate the equipment and interpret theresults. Specimens may analyzed to determine the presence of one or moreanalytes within the specimen, e.g., a pathogen or virus, a particularchemical, and antigen or antibody, etc, for medical purposes. In somecases, bioassays are performed by tagging a sample with a fluorescenttag that emit light of a particular wavelength. The tag may beilluminated with an excitation light source to cause fluorescence. Thefluorescence is detected with a photodetector, and the signal analyzedto determine a property about the sample. Bioassays using fluorescenttags conventionally involve expensive laser light sources and opticsarranged to illuminate samples. The assays may further involve bulky,expensive collection optics arranged to collect the fluorescence fromthe samples as well as expensive electronic instrumentation to processthe signals.

Because conventional analytical equipment is typically expensive andrequires a skilled operator, specimens to be analyzed may need to besent to an on-site or off-site facility for processing. This canintroduce appreciable delay and cost associated with even routineanalysis of a specimen. For example, a patient may have to wait severaldays and schedule a return visit to a doctor's office to learn about theresults of a laboratory test on a specimen provided by the patient.

SUMMARY

The technology described herein relates to apparatus and methods foranalyzing specimens rapidly using an active-source-pixel, integrateddevice that can be interfaced with a mobile computing instrument. Theintegrated device may be in the form of a disposable or recyclablelab-on-chip or a packaged module that is configured to receive a smallamount of a specimen and execute, in parallel, a large number ofanalyses of samples within the specimen. The integrated device may beused to detect the presence of particular chemical or biologicalanalytes in some embodiments, to evaluate a chemical or biologicalreactions in some embodiments, and to determine genetic sequences insome embodiments. According to some implementations, the integrateddevice may be used for single-molecule gene sequencing.

According to some implementations, a user deposits a specimen in achamber on the integrated device, and inserts the integrated device intoa receiving instrument. The receiving instrument, alone or incommunication with a computer, automatically interfaces with theintegrated device, receives data from the integrated device, processesthe received data, and provides results of the analysis to the user. Asmay be appreciated, integration and computing intelligence on the chip,receiving instrument, and or computer reduce the skill level requiredfrom the user.

Embodiments include methods for sequencing nucleic acid molecules.According to some embodiments, a first method of sequencing a nucleicacid molecule may comprise providing excitation energy to a sample wellformed at a first pixel on a substrate, and receiving, at a sensorformed at the first pixel, a first emission from the sample well,wherein the first emission is associated with a type of nucleic acidsubunit from among different types of nucleic acid subunits. The methodmay further include producing, by the sensor, a first signal and asecond signal representative of the received first emission, analyzingthe first signal and the second signal, and identifying the type of thenucleic acid subunit based upon the analysis of the first signal and thesecond signal.

According to some embodiments, a second method for sequencing a targetnucleic acid molecule may comprise providing a integrated device thatincludes (i) a sample well containing said target nucleic acid molecule,a polymerizing enzyme and a plurality of types of nucleotides ornucleotide analogs, and (ii) at least one excitation source that directsexcitation energy to said sample well, and performing an extensionreaction at a priming location of said target nucleic acid molecule inthe presence of said polymerizing enzyme to sequentially incorporatesaid nucleotides or nucleotide analogs into a growing strand that iscomplementary to said target nucleic acid molecule, wherein uponincorporation and excitation by excitation energy from said excitationsource, said nucleotides or nucleotide analogs produce emissions fromsaid sample well. The second method may further include detecting saidemissions at a sensor that is configured to receive said emissions fromsaid sample well, receiving signal sets from the sensor for eachdetected emission, wherein the signal sets are representative of spatialand/or temporal distributions of said detected emissions and distinguishtypes of nucleotides or nucleotide analogs, and identifying the types ofnucleotides or nucleotide analogs based on said received signal sets,thereby sequencing said target nucleic acid molecule.

Various embodiments of integrated devices are contemplated. According tosome embodiments, an integrated device for analyzing a plurality ofsamples in parallel may comprise a plurality of pixels arranged on asubstrate, wherein an individual pixel of the plurality of pixelscomprises (i) a sample well having an excitation region configured toretain a biological sample, (ii) a first structure located adjacent toor within the sample well and configured to affect coupling of at leastexcitation energy into the excitation region, and (iii) a sensing systemincluding a sensor that is configured to discriminate between at leasttwo different emissions from the sample well, wherein the two differentemissions comprise spectral and/or temporal differences. The firststructure may additionally affect coupling of emission from the samplewell to the sensor. The integrated device may further include at leastone excitation source on the substrate that is arranged to provide theexcitation energy to the sample well, and circuitry arranged on thesubstrate to receive at least one signal from the sensor.

In some embodiments, an integrated device may comprise a plurality ofsample wells, excitation sources, and sensors having any suitablecombination of the foregoing features and functionalities. Further, theplurality of sample wells, excitation sources, and sensors on anintegrated device may be substantially identical for some embodiments ofan integrated device, whereas in other embodiments, the sample wells,excitation sources, and sensors may differ across an integrated device.For example, there may be groups of sample wells, excitation sources,and sensors on an integrated device, each group having a distinguishablesubset of the foregoing features and functionalities associated with asample well, excitation source, and/or sensor.

Instruments are also contemplated that may be configured to receive andcommunicated with an integrated device. According to some embodiments, aportable instrument configured to receive and communicate with anintegrated device may comprise at least one processor, a dock configuredto receive an integrated device as described in any of the aboveembodiments, a cover configured to exclude a majority external lightfrom entering the dock, and a first plurality of electrical contactsconfigured to connect to a second plurality of electrical contacts onthe integrated device, wherein power may be provide to the integrateddevice through at least some of the first plurality of electricalcontacts and the at least one signal from each sensor may be receivedthrough at least some of the first plurality of electrical contacts. Insome implementations, the first plurality of electrical contacts isformed on a user-removable interposer. In some implementations, thefirst plurality of electrical contacts is configured to contact a thirdplurality of contacts on a user-replaceable interposer. According tosome embodiments, a the portable instrument further comprises acommunication interface, wherein the communication interface comprises aUSB interface, a Thunderbolt interface, or a high-speed digitalinterface.

According to some embodiments, a third method of analyzing a pluralityof samples in parallel may comprise receiving, at a surface of asubstrate, a specimen containing samples, retaining, in a plurality ofsample wells located in a plurality of pixels on the substrate, samplesfrom the fluid suspension, and providing excitation energy to one ormore of the sample wells from at least one excitation source. The thirdmethod may further include, at least for one of the plurality of pixels,detecting an emission from a sample well at a sensor that is arranged toreceive emission from the sample well, receiving a signal set from thesensor representative of the detected emission, and identifying aproperty of a sample retained in the sample well based on an analysis ofthe signal set.

Methods associated with fabrication of an integrated device are alsocontemplated. According to some embodiments, a first method forfabricating a sample well and optical structure aligned to the samplewell may comprise acts of forming, in a same patterning step, a patternfor the sample well and for the optical structure in a first resistlayer disposed on a substrate, covering at least the pattern of thesample well with a second resist layer, etching a pattern of the opticalstructure into the substrate, removing portions of the first resistlayer not covered by the second resist layer, removing the second resistlayer, depositing a material over the substrate, and removing theremaining portion of the first resist layer.

According to some embodiments, a second method for fabricating a samplewell may comprise forming, in a same patterning step, a pattern for thesample well and for the optical structure in a first layer disposed on asubstrate, etching the pattern of the sample well and the opticalstructure into the substrate, covering at least the pattern of thesample well with a resist layer, depositing a material over thesubstrate, wherein the material fills voids etched into the substratefrom the etching of the pattern of the optical structure, and removingthe resist layer. In some implementations, the first layer comprises aconductive material. In some aspects, the optical structure comprises acircular grating. In some implementations, the substrate is opticallytransparent. According to some implementations, removing the resistlayer leaves a sample well having a transverse dimension less than 500nm and including a divot at a bottom of the sample well etched into thesubstrate.

Methods for fabricating excitation sources are also contemplated.According to some embodiments, a method of forming a nano-scaleexcitation source aligned to a sample well may comprise etching a viainto an insulating layer of a substrate, the substrate comprising asemiconductor layer, an insulating layer adjacent the semiconductorlayer, and a first conductive layer adjacent the insulating layer,forming a sacrificial coating on walls of the via, etching the via tothe semiconductor layer, and epitaxially growing a semiconductor pillarhaving a first conductivity type within the via from the semiconductorlayer. In some implementations, the method may further comprise removingthe sacrificial coating to expose walls of the pillar at a portion ofthe pillar, epitaxially growing a semiconductor layer having a secondconductivity type over the portion of the pillar, and conformallydepositing a second conductive layer over the semiconductor layer,wherein the second conductive layer electrically connects to the firstconductive layer. In some aspects, the semiconductor pillar andsemiconductor layer comprise a light-emitting diode or laser diode. Insome aspects, the semiconductor pillar and semiconductor layer comprisea semiconductor diode. In some implementations, an end of theepitaxially-grown semiconductor pillar nearest the first conductivelayer lies a distance from a nearest surface of the first conductivelayer. In some implementations, an unfilled region of the via forms asample well. According to some implementations, a transverse dimensionof the semiconductor pillar is less than 200 nm. In some aspects, theinsulating layer is optically transparent.

The foregoing features and acts associated with aspects andimplementations of the method for forming an excitation source may beincluded in any suitable combination in one or more embodiments of amethod for forming an excitation source.

Although the foregoing methods and devices may be described in referenceto a single element (e.g., a sample well, an excitation source, asensor, an excitation-coupling structure, an emission-couplingstructure), the methods may be implemented in parallel to fabricate alarge number of devices in parallel (e.g., using micro- andnano-fabrication processes). Further, the devices may be arranged in alarge number on an integrated device.

The term “pixel” may be used in the present disclosure to refer to aunit cell of an integrated device. The unit cell may include a samplewell and a sensor. The unit cell may further include an excitationsource. The unit cell may further include at least oneexcitation-coupling optical structure (which may be referred to as a“first structure”) that is configured to enhance coupling of excitationenergy from the excitation source to the sample well. The unit cell mayfurther include at least one emission-coupling structure that isconfigured to enhance coupling of emission from the sample well to thesensor. The unit cell may further include integrated electronic devices(e.g., CMOS devices). There may be a plurality of pixels arranged in anarray on an integrated device.

The term “optical” may be used in the present disclosure to refer tovisible, near infrared, and short-wavelength infrared spectral bands.

The term “tag” may be used in the present disclosure to refer to a tag,probe, marker, or reporter attached to a sample to be analyzed orattached to a reactant that may be reacted with a sample.

The phrase “excitation energy” may be used in the present disclosure torefer to any form of energy (e.g., radiative or non-radiative) deliveredto a sample and/or tag within the sample well. Radiative excitationenergy may comprise optical radiation at one or more characteristicwavelengths.

The phrase “characteristic wavelength” may be used in the presentdisclosure to refer to a central or predominant wavelength within alimited bandwidth of radiation. In some cases, it may refer to a peakwavelength of a bandwidth of radiation. Examples of characteristicwavelengths of fluorophores are 563 nm, 595 nm, 662 nm, and 687 nm.

The phrase “characteristic energy” may be used in the present disclosureto refer to an energy associated with a characteristic wavelength.

The term “emission” may be used in the present disclosure to refer toemission from a tag and/or sample. This may include radiative emission(e.g., optical emission) or non-radiative energy transfer (e.g., Dexterenergy transfer or Forster resonant energy transfer). Emission resultsfrom excitation of a sample and/or tag within the sample well.

The phrase “emission from a sample well” or “emission from a sample” maybe used in the present disclosure to refer to emission from a tag and/orsample within a sample well. The phrase “emission from a sample well”may also be used in the present disclosure to refer to emission from acalibration particle (e.g., a fluorescent polystyrene bead, a quantumdot, etc.) within a sample well.

The term “self-aligned” may be used in the present disclosure to referto a microfabrication process in which at least two distinct elements(e.g., a sample well and an emission-coupling structure, a sample welland an excitation-source) may be fabricated and aligned to each otherwithout using two separate lithographic patterning steps in which afirst lithographic patterning step (e.g., photolithography, ion-beamlithography, EUV lithography) prints a pattern of a first element and asecond lithographic patterning step is aligned to the first lithographicpatterning step and prints a pattern of the second element. Aself-aligned process may comprise including the pattern of both thefirst and second element in a single lithographic patterning step, ormay comprise forming the second element using features of a fabricatedstructure of the first element.

The term “sensor” may be used in the present disclosure to refer to oneor more integrated circuit devices configured to sense emission from thesample well and produce at least one electrical signal representative ofthe sensed emission.

The term “nano-scale” may be used in the present disclosure to refer toa structure having at least one dimension or minimum feature size on theorder of 150 nanometers (nm) or less, but not greater than approximately500 nm.

The term “micro-scale” may be used in the present disclosure to refer toa structure having at least one dimension or minimum feature sizebetween approximately 500 nm and approximately 100 microns.

The term “nanoaperture” may be used in the present disclosure to referto a nano-scale opening or aperture in at least one layer of material.For example, a diameter or width of the opening is less thanapproximately 500 nm.

The term “nanohole” may be used in the present disclosure to refer to anano-scale hole formed in at least one layer of material. A nanohole mayhave a length or longitudinal dimension that is greater than ananoaperture.

The term “sub-cutoff nanoaperture” may be used in the present disclosureto refer to a waveguide structure that does not support a propagatingmode for a selected wavelength of radiation that may be incident on thewaveguide structure. For example, the selected wavelength may be longerthan a cut-off wavelength for the waveguide structure, and power decaysexponentially into the waveguide.

The phrase “enhance excitation energy” may be used in the presentdisclosure to refer to increasing an intensity of excitation energy atan excitation region of a sample well. The intensity may be increased byconcentrating and/or resonating excitation energy incident on the samplewell, for example. In some cases, the intensity may be increased byanti-reflective coatings or lossy layers that allow the excitationenergy to penetrate further into the excitation region of a sample well.An enhancement of excitation energy may be a comparative reference to anembodiment that does not include structures to enhance the excitationenergy at an excitation region of a sample well.

The terms “about,” “approximately,” and “substantially” may be used inthe present disclosure to refer to a value, and are intended toencompass the referenced value plus and minus acceptable variations. Theamount of variation could be less than 5% in some embodiments, less than10% in some embodiments, and yet less than 20% in some embodiments. Inembodiments where an apparatus may function properly over a large rangeof values, e.g., a range including one or more orders of magnitude, theamount of variation could be a factor of two. For example, if anapparatus functions properly for a value ranging from 20 to 350,“approximately 80” may encompass values between 40 and 160.

The term “adjacent” may be used in the present disclosure to refer totwo elements arranged within close proximity to one another (e.g.,within a distance that is less than about one-fifth of a transverse orvertical dimension of a pixel). In some cases there may be interveningstructures or layers between adjacent elements. I some cases adjacentelements may be immediately adjacent to one another with no interveningstructures or elements.

The term “detect” may be used in the present disclosure to refer toreceiving an emission at a sensor from a sample well and producing atleast one electrical signal representative of or associated with theemission. The term “detect” may also be used in the present disclosureto refer to determining the presence of, or identifying a property of, aparticular sample or tag in the sample well based upon emission from thesample well.

The foregoing and other aspects, implementations, acts, functionalities,features and, embodiments of the present teachings can be more fullyunderstood from the following description in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that in someinstances various aspects of the invention may be shown exaggerated orenlarged to facilitate an understanding of the invention. In thedrawings, like reference characters generally refer to like features,functionally similar and/or structurally similar elements throughout thevarious figures. The drawings are not necessarily to scale, emphasisinstead being placed upon illustrating the principles of the teachings.The drawings are not intended to limit the scope of the presentteachings in any way.

FIG. 1-1 depicts emission wavelength spectra, according to someembodiments.

FIG. 1-2A depicts absorption wavelength spectra, according to someembodiments.

FIG. 1-2B depicts emission wavelength spectra, according to someembodiments.

FIG. 2-1 is a block diagram representation of a compact apparatus thatmay be used for rapid, mobile analysis of biological and chemicalspecimens, according to some embodiments.

FIG. 2-2 depicts and integrated device, according to some embodiments.

FIG. 2-3 depicts a computing system, according to some embodiments.

FIG. 3-1 depicts a sample well formed in a pixel region of an integrateddevice, according to one embodiment.

FIG. 3-2 depicts excitation energy incident on a sample well, accordingto some embodiments.

FIG. 3-3 illustrates attenuation of excitation energy along a samplewell that is formed as a zero-mode waveguide, according to someembodiments.

FIG. 3-4 depicts a sample well that includes a divot, which increasesexcitation energy at an excitation region associated with the samplewell in some embodiments.

FIG. 3-5 compares excitation intensities for sample wells with andwithout a divot, according to one embodiment.

FIG. 3-6 depicts a sample well and divot formed at a protrusion,according to some embodiments.

FIG. 3-7A depicts a sample well having tapered sidewalls, according tosome embodiments.

FIG. 3-7B depicts a sample well having curved sidewalls and a divot witha smaller transverse dimension, according to some embodiments.

FIG. 3-7C and FIG. 3-7D depict a sample well formed from surfaceplasmonic structures.

FIG. 3-7E depicts a sample well that includes anexcitation-energy-enhancing structure formed along sidewalls of thesample well, according to some embodiments.

FIG. 3-7F depicts a sample well formed in a multi-layer stack, accordingto some embodiments.

FIG. 3-8 illustrates surface coating formed on surfaces of a samplewell, according to some embodiments.

FIG. 3-9A through FIG. 3-9E depict structures associated with a lift-offprocess of forming a sample well, according to some embodiments.

FIG. 3-9F depicts a structure associated with an alternative lift-offprocess of forming a sample well, according to some embodiments.

FIG. 3-10A through FIG. 3-10D depict structures associated with a directetching process of forming a sample well, according to some embodiments.

FIG. 3-11 depicts a sample well that may be formed in multiple layersusing a lift-off process or a direct etching process, according to someembodiments.

FIG. 3-12 depicts a structure associated with an etching process thatmay be used to form a divot, according to some embodiments.

FIG. 3-13A through FIG. 3-13C depict structures associated with analternative process of forming a divot, according to some embodiments.

FIG. 3-14A through FIG. 3-14D depict structures associated with aprocess for depositing an adherent and passivating layers, according tosome embodiments.

FIG. 3-15 depicts a structure associated with a process for depositingan adherent centrally within a sample well, according to someembodiments.

FIG. 4-1A and FIG. 4-1B depict spectral excitation bands of excitationsources, according to some embodiments.

FIG. 4-2A through FIG. 4-2D depict, in plan view, various arrangementsof excitation sources that may be included on an integrated device,according to some implementations.

FIG. 4-2E depicts, in elevation view, an arrangement of an excitationsource located adjacent to a pixel region, according to someembodiments.

FIG. 4-3A depicts an organic light emitting diode (OLED) integratedwithin a pixel, according to some embodiments.

FIG. 4-3B depicts further details of a light emitting diode structureintegrated within a pixel, according to some embodiments.

FIG. 4-3C depicts a vertical cavity surface emitting laser (VCSEL)integrated within a pixel, according to some embodiments.

FIG. 4-3D depicts a self-aligned nano-LED integrated within a pixel,according to some embodiments.

FIG. 4-3E depicts a self-aligned nano-VCSEL integrated within a pixel,according to some embodiments.

FIG. 4-4A through FIG. 4-4F depict structures associated with processsteps for fabricating a nano-LED or nano-VCSEL, according to someembodiments.

FIG. 4-4G through FIG. 4-4I depict structures associated withalternative process steps for fabricating a nano-LED, according to someembodiments.

FIG. 4-5A depicts a non-radiative excitation source that may beintegrated in a pixel, according to some embodiments.

FIG. 4-5B depicts, in elevation view, a non-radiative excitation sourcethat may be integrated in a pixel, according to some embodiments.

FIG. 4-5C depicts, in plan view, interconnects for a non-radiativeexcitation source, according to some embodiments.

FIG. 4-5D depicts a nano-diode, non-radiative excitation source that maybe integrated in a pixel, according to some embodiments.

FIG. 4-6A through FIG. 4-6U depict structures associated with processsteps for fabricating a self-aligned, non-radiative excitation sources,according to some embodiments.

FIG. 5-1A and FIG. 5-1B depict a surface-plasmon structure, according tojust one embodiment.

FIG. 5-1C depicts a surface-plasmon structure formed adjacent a samplewell, according to some embodiments.

FIG. 5-1D and FIG. 5-1E depict surface-plasmon structures formed in asample well, according to some embodiments.

FIG. 5-2A through FIG. 5-2C depict examples of periodic surface-plasmonstructures, according to some embodiments.

FIG. 5-2D depicts a numerical simulation of excitation radiation at asample well formed adjacent a periodic surface-plasmon structure,according to some embodiments.

FIG. 5-2E through FIG. 5-2G depict periodic surface-plasmon structures,according to some embodiments.

FIG. 5-2H and FIG. 5-2I depict a nano-antenna comprising surface-plasmonstructures, according to some embodiments.

FIG. 5-3A through FIG. 5-3E depict structures associated with processsteps for forming a surface-plasmon structure, according to someembodiments.

FIG. 5-4A through FIG. 5-4G depict structures associated with processsteps for forming a surface-plasmon structure and self-aligned samplewell, according to some embodiments.

FIG. 5-5A through FIG. 5-5E depict structures associated with processsteps for forming a surface-plasmon structure and self-aligned samplewell, according to some embodiments.

FIG. 5-6A depicts a thin lossy film formed adjacent a sample well,according to some embodiments.

FIG. 5-6B and FIG. 5-6C depict results from numerical simulations ofexcitation radiation in the vicinity of a sample well and thin lossyfilm, according to some embodiments.

FIG. 5-6D depicts a thin lossy film spaced from a sample well, accordingto some embodiments.

FIG. 5-6E depicts a thin lossy film stack formed adjacent a sample well,according to some embodiments.

FIG. 5-7A illustrates a reflective stack that may be used to form aresonant cavity adjacent a sample well, according to some embodiments.

FIG. 5-7B depicts a dielectric structure that may be used to concentrateexcitation radiation at a sample well, according to some embodiments.

FIG. 5-7C and FIG. 5-7D depict a photonic bandgap structure that may bepatterned adjacent a sample well, according to some embodiments.

FIG. 5-8A through FIG. 5-8G depict structures associated with processsteps for forming dielectric structures and a self-aligned sample well,according to some embodiments.

FIG. 5-9A and FIG. 5-9B depict structures for coupling excitation energyto a sample via a non-radiative process, according to some embodiments.

FIG. 5-9C and FIG. 5-9D depicts a structure for coupling excitationenergy to a sample by multiple non-radiative processes, according tosome embodiments.

FIG. 5-9E depicts a structure that incorporates one or moreenergy-converting particles to couple excitation energy to a sample viaa radiative or non-radiative process, according to some embodiments.

FIG. 5-9F depicts spectra associated with down conversion of excitationenergy to a sample, according to some embodiments.

FIG. 5-9G depicts spectra associated with up conversion of excitationenergy to a sample, according to some embodiments.

FIG. 6-1A depicts a concentric, plasmonic circular grating, according tosome embodiments.

FIG. 6-1B depicts a spiral plasmonic grating, according to someembodiments.

FIG. 6-2A through FIG. 6-2D depict emission spatial distributionpatterns from a concentric, plasmonic circular grating for variousemission wavelengths, according to some embodiments.

FIG. 6-3A through FIG. 6-3D depict plasmonic nano-antennas, according tosome embodiments.

FIG. 6-4A depicts a pattern for a spiral, plasmonic nano-antenna,according to some embodiments.

FIG. 6-4B depicts results from a numerical simulation of electromagneticfield in the vicinity of the spiral, plasmonic nano-antenna of FIG.6-4A, according to some embodiments.

FIG. 6-4C through FIG. 6-4E illustrate various configurations of spiral,plasmonic nano-antennas, according to some embodiments.

FIG. 6-5A through FIG. 6-5D depicts results from numerical simulationsof spatial distribution patterns associated with different wavelengthsthat emit from a sample well surrounded by a plasmonic nano-antenna,according to some embodiments.

FIG. 6-6A and FIG. 6-6B depicts far-field spectral sorting optics,according to some embodiments.

FIG. 6-7A and FIG. 6-7B depicts far-field spectral filtering optics,according to some embodiments.

FIG. 7-1A depicts, in elevation view, a sensor 3-260 within a pixel,according to some embodiments.

FIG. 7-1B depicts a bulls-eye sensor having two separate and concentricactive areas, according to some embodiments.

FIG. 7-1C depicts a stripe sensor having four separate active areas,according to some embodiments.

FIG. 7-1D depicts a quad sensor having four separate active areas,according to some embodiments.

FIG. 7-1E depicts an arc-segment sensor having four separate activeareas, according to some embodiments.

FIG. 7-1F depicts a stacked-segment sensor, according to someembodiments.

FIG. 7-2A depicts an emission distribution from a sample well forradiation emitted at a first wavelength, according to some embodiments.

FIG. 7-2B depicts a radiation pattern received by a bulls-eye sensorcorresponding to the emission distribution depicted in FIG. 7-2A,according to some embodiments.

FIG. 7-2C depicts an emission distribution from a sample well forradiation emitted at a second wavelength, according to some embodiments.

FIG. 7-2D depicts a radiation pattern received by a bulls-eye sensorcorresponding to the emission distribution depicted in FIG. 7-2C,according to some embodiments.

FIG. 7-2E represents results from a numerical simulation of signaldetection for a bulls-eye sensor having two active areas for a firstemission wavelength from a sample, according to some embodiments.

FIG. 7-2F represents results from a numerical simulation of signaldetection for the bulls-eye sensor associated with FIG. 7-2E for asecond emission wavelength from a sample, according to some embodiments.

FIG. 7-2G represents results from a numerical simulation of signaldetection for the bulls-eye sensor associated with FIG. 7-2E for a thirdemission wavelength from a sample, according to some embodiments.

FIG. 7-2H represents results from a numerical simulation of signaldetection for the bulls-eye sensor associated with FIG. 7-2E for afourth emission wavelength from a sample, according to some embodiments.

FIG. 7-2I represents results from a numerical simulation of signaldetection for a bulls-eye sensor having four active areas for a firstemission wavelength from a sample, according to some embodiments.

FIG. 7-2J represents results from a numerical simulation of signaldetection for the bulls-eye sensor associated with FIG. 7-2I for asecond emission wavelength from a sample, according to some embodiments.

FIG. 7-3A depicts circuitry on an integrated device that may be used toread signals from a sensor comprising two active areas, according tosome embodiments.

FIG. 7-3B depicts a three-transistor circuit that may be included at asensor segment for signal accumulation and read-out, according to someembodiments.

FIG. 7-3C depicts circuitry on an integrated device that may be used toread signals from a sensor comprising four active areas, according tosome embodiments.

FIG. 7-4A depicts temporal emission characteristics for two differentemitters that may be used for sample analysis, according to someembodiments.

FIG. 7-4B depicts temporal evolution of an excitation source andluminescence from a sample, according to some embodiments.

FIG. 7-4C illustrates time-delay sampling, according to someembodiments.

FIG. 7-4D depicts temporal emission characteristics for two differentemitters, according to some embodiments.

FIG. 7-4E depicts voltage dynamics at a charge-accumulation node of asensor, according to some embodiments.

FIG. 7-4F depicts a double read of a sensor segment without reset,according to some embodiments.

FIG. 7-4G and FIG. 7-4H illustrate first and second read signal levelsassociated with two emitters having temporally-distinct emissioncharacteristics, according to some embodiments.

FIG. 8-1 depicts a method of operation of a compact apparatus that maybe used for rapid, mobile analysis of biological and chemical specimens,according to some embodiments.

FIG. 8-2 depicts a calibration procedure, according to some embodiments.

FIG. 8-3 depicts a data-analysis procedure, according to someembodiments.

The features and advantages of the present invention will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings.

When describing embodiments in reference to the drawings, directionreferences (“above,” “below,” “top,” “bottom,” “left,” “right,”“horizontal,” “vertical,” etc.) may be used. Such references areintended merely as an aid to the reader viewing the drawings in a normalorientation. These directional references are not intended to describe apreferred or only orientation of an embodied device. A device may beembodied in other orientations.

DETAILED DESCRIPTION

I. Introduction

The inventors have recognized and appreciated that conventionalapparatuses for performing bioassays are large, expensive, and requireadvanced laboratory techniques to perform. The inventors have recognizedand appreciated that there is a need for a compact device that cansimply and inexpensively analyze biological and/or chemical specimensfor medical, forensic, research, and various diagnostic purposes. Anapplication of such device may be for sequencing a biomolecule, such asa nucleic acid molecule or a polypeptide (e.g., protein) having aplurality of amino acids. A compact, high-speed apparatus for performingdetection and quantitation of single molecules or particles could reducethe cost of performing complex quantitative measurements of biologicaland/or chemical samples and rapidly advance the speed of research anddevelopment in various fields of biochemistry. Moreover, acost-effective device that is readily transportable could transform notonly the way bioassays are performed in the developed world, but providepeople in developing regions, for the first time, ready access toessential diagnostic tests that could dramatically improve their healthand well-being. For example, in some embodiments, an apparatus forperforming bioassays is used to perform diagnostic tests of biologicalsamples, such as blood, urine and/or saliva that may be used byindividuals in their home, by a doctor in the field, or at a remoteclinic in developing countries or any other location, such as ruraldoctors' offices. Such diagnostic tests can include the detection ofbiomolecules in a biological sample of a subject, such as a nucleic acidmolecule or protein. In some examples, diagnostic tests includesequencing a nucleic acid molecule in a biological sample of a subject,such as sequencing of cell free deoxyribonucleic acid molecules orexpression products in a biological sample of the subject.

Although a compact instrument may be used for detecting the presence ofbiochemical species (e.g., nucleic acids, proteins, antigens,antibodies, viruses, small molecules, etc.) in specimens or biologicalsamples, the instrument may be used for more complicated tasks, such asanalyzing dynamic biochemical processes. One field of interest in whichthe instrument may be used is single-molecule genetic sequencing.According to some embodiments, real-time, single-molecule nucleic acidsequencing may be performed with the instrument to decode genes or genesegments. This may allow clinicians, for example, to track mutations ofharmful viruses in real time.

The term “nucleic acid,” as used herein, generally refers to a moleculecomprising one or more nucleic acid subunits. A nucleic acid may includeone or more subunits selected from adenosine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), or variants thereof. In some examples,a nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),or derivatives thereof. A nucleic acid may be single-stranded or doublestranded. A nucleic acid may be circular.

The term “nucleotide,” as used herein, generally refers to a nucleicacid subunit, which can include A, C, G, T or U, or variants or analogsthereof. A nucleotide can include any subunit that can be incorporatedinto a growing nucleic acid strand. Such subunit can be an A, C, G, T,or U, or any other subunit that is specific to one or more complementaryA, C, G, T or U, or complementary to a purine (i.e., A or G, or variantor analogs thereof) or a pyrimidine (i.e., C, T or U, or variant oranalogs thereof). A subunit can enable individual nucleic acid bases orgroups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, oruracil-counterparts thereof) to be resolved.

A nucleotide generally includes a nucleoside and at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more phosphate (PO₃) groups. A nucleotide can includea nucleobase, a five-carbon sugar (either ribose or deoxyribose), andone or more phosphate groups. Ribonucleotides are nucleotides in whichthe sugar is ribose. Deoxyribonucleotides are nucleotides in which thesugar is deoxyribose. A nucleotide can be a nucleoside monophosphate ora nucleoside polyphosphate. A nucleotide can be a deoxyribonucleosidepolyphosphate, such as, e.g., a deoxyribonucleoside triphosphate, whichcan be selected from deoxyadenosine triphosphate (dATP), deoxycytidinetriphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyuridinetriphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTPs, thatinclude detectable tags, such as luminescent tags or markers (e.g.,fluorophores).

A nucleoside polyphosphate can have ‘n’ phosphate groups, where ‘n’ is anumber that is greater than or equal to 2, 3, 4, 5, 6, 7, 8, 9, or 10.Examples of nucleoside polyphosphates include nucleoside diphosphate andnucleoside triphosphate. A nucleotide can be a terminal phosphatelabeled nucleoside, such as a terminal phosphate labeled nucleosidepolyphosphate. Such label can be a luminescent (e.g., fluorescent orchemiluminescent) label, a fluorogenic label, a colored label, achromogenic label, a mass tag, an electrostatic label, or anelectrochemical label. A label (or marker) can be coupled to a terminalphosphate through a linker. The linker can include, for example, atleast one or a plurality of hydroxyl groups, sulfhydryl groups, aminogroups or haloalkyl groups, which may be suitable for forming, forexample, a phosphate ester, a thioester, a phosphoramidate or an alkylphosphonate linkage at the terminal phosphate of a natural or modifiednucleotide. A linker can be cleavable so as to separate a label from theterminal phosphate, such as with the aid of a polymerization enzyme.Examples of nucleotides and linkers are provided in U.S. Pat. No.7,041,812, which is entirely incorporated herein by reference.

The term “polymerase,” as used herein, generally refers to any enzyme(or polymerizing enzyme) capable of catalyzing a polymerizationreaction. Examples of polymerases include, without limitation, a nucleicacid polymerase, a transcriptase or a ligase. A polymerase can be apolymerization enzyme.

The term “genome” generally refers to an entirety of an organism'shereditary information. A genome can be encoded either in DNA or in RNA.A genome can comprise coding regions that code for proteins as well asnon-coding regions. A genome can include the sequence of all chromosomestogether in an organism. For example, the human genome has a total of 46chromosomes. The sequence of all of these together constitutes the humangenome.

The present disclosure provides devices, systems and methods fordetecting biomolecules or subunits thereof, such as nucleic acidmolecules. Such detection can include sequencing. A biomolecule may beextracted from a biological sample obtained from a subject. Thebiological sample may be extracted from a bodily fluid or tissue of thesubject, such as breath, saliva, urine or blood (e.g., whole blood orplasma). The subject may be suspected of having a health condition, suchas a disease (e.g., cancer). In some examples, one or more nucleic acidmolecules are extracted from the bodily fluid or tissue of the subject.The one or more nucleic acids may be extracted from one or more cellsobtained from the subject, such as part of a tissue of the subject, orobtained from a cell-free bodily fluid of the subject, such as wholeblood.

A biological sample may be processed in preparation for detection (e.g.,sequencing). Such processing can include isolation and/or purificationof the biomolecule (e.g., nucleic acid molecule) from the biologicalsample, and generation of more copies of the biomolecule. In someexamples, one or more nucleic acid molecules are isolated and purifiedform a bodily fluid or tissue of the subject, and amplified throughnucleic acid amplification, such as polymerase chain reaction (PCR).Then, the one or more nucleic acids molecules or subunits thereof can beidentified, such as through sequencing.

Sequencing can include the determination of individual subunits of atemplate biomolecule (e.g., nucleic acid molecule) by synthesizinganother biomolecule that is complementary or analogous to the template,such as by synthesizing a nucleic acid molecule that is complementary toa template nucleic acid molecule and identifying the incorporation ofnucleotides with time (i.e., sequencing by synthesis). As analternative, sequencing can include the direct identification ofindividual subunits of the biomolecule.

During sequencing, signals indicative of individual subunits of abiomolecule may be collected in memory and processed in real time or ata later point in time to determine a sequence of the biomolecule. Suchprocessing can include a comparison of the signals to reference signalsthat enable the identification of the individual subunits, which in somecases yields reads. Reads may be sequences of sufficient length (e.g.,at least about 30 base pairs (bp)) that can be used to identify a largersequence or region, e.g., that can be aligned to a location on achromosome or genomic region or gene.

Sequence reads can be used to reconstruct a longer region of a genome ofa subject (alignment). Reads can be used to reconstruct chromosomalregions, whole chromosomes, or the whole genome. Sequence reads or alarger sequence generated from such reads can be used to analyze agenome of a subject, such as identify variants or polymorphisms.Examples of variants include, but are not limited to, single nucleotidepolymorphisms (SNPs) including tandem SNPs, small-scale multi-basedeletions or insertions, also referred to as indels or deletioninsertion polymorphisms or DIPs), Multi-Nucleotide Polymorphisms (MNPs),Short Tandem Repeats (STRs), deletions, including microdeletions,insertions, including microinsertions, structural variations, includingduplications, inversions, translocations, multiplications, complexmulti-site variants, copy number variations (CNV). Genomic sequences cancomprise combinations of variants. For example, genomic sequences canencompass the combination of one or more SNPs and one or more CNVs.

Individual subunits of biomolecules may be identified using markers. Insome examples, luminescent markers are used to identify individualsubunits of biomolecules, as described elsewhere herein.

Nucleic acid (e.g., DNA) sequencing allows for the determination of theorder and position of nucleotides in a target nucleic acid molecule.Nucleic acid sequencing technologies may vary in the methods used todetermine the nucleic acid sequence as well as in the rate, read length,and incidence of errors in the sequencing process. For example, somenucleic acid sequencing methods are based on sequencing by synthesis, inwhich the identity of a nucleotide is determined as the nucleotide isincorporated into a newly synthesized strand of nucleic acid that iscomplementary to the target nucleic acid molecule.

During sequencing, a polymerizing enzyme may couple (e.g., attach) to apriming location of a target nucleic acid molecule. The priming locationcan be a primer that is complementary to the target nucleic acidmolecule. As an alternative the priming location is a gap or nick thatis provided within a double stranded segment of the target nucleic acidmolecule. A gap or nick can be from 0 to at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, or 40 nucleotides in length. A nick can provide abreak in one strand of a double stranded sequence, which can provide apriming location for a polymerizing enzyme, such as, for example, astrand displacing polymerase enzyme.

In some cases, a sequencing primer can be annealed to a target nucleicacid molecule that may or may not be immobilized to a solid support,such as a sample well. In some embodiments, a sequencing primer may beimmobilized to a solid support and hybridization of the target nucleicacid molecule also immobilizes the target nucleic acid molecule to thesolid support. Via the action of an enzyme (e.g., a polymerase) capableof adding or incorporating a nucleotide to the primer, nucleotides canbe added to the primer in 5′ to 3′, template bound fashion. Suchincorporation of nucleotides to a primer (e.g., via the action of apolymerase) can generally be referred to as a primer extension reaction.Each nucleotide can be associated with a detectable tag that can bedetected and used to determine each nucleotide incorporated into theprimer and, thus, a sequence of the newly synthesized nucleic acidmolecule. Via sequence complementarity of the newly synthesized nucleicacid molecule, the sequence of the target nucleic acid molecule can alsobe determined. In some cases, annealing of a sequencing primer to atarget nucleic acid molecule and incorporation of nucleotides to thesequencing primer can occur at similar reaction conditions (e.g., thesame or similar reaction temperature) or at differing reactionconditions (e.g., different reaction temperatures). Moreover, somesequencing by synthesis methods can include the presence of a populationof target nucleic acid molecules (e.g., copies of a target nucleic acid)and/or a step of amplification of the target nucleic acid to achieve apopulation of target nucleic acids.

Devices and systems of the present disclosure are capable of sequencingsingle nucleic acid molecules with high accuracy and long read lengths,such as an accuracy of at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999%, and/orread lengths greater than or equal to about 10 base pairs (bp), 50 bp,100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 1000 bp, 10,000 bp, 20,000 bp,30,000 bp, 40,000 bp, 50,000 bp, or 100,000 bp. In some embodiments, thetarget nucleic acid molecule used in single molecule sequencing is asingle-stranded target nucleic acid (e.g., deoxyribonucleic acid (DNA),DNA derivatives, ribonucleic acid (RNA), RNA derivatives) template thatis added or immobilized to a sample well containing at least oneadditional component of a sequencing reaction (e.g., a polymerase suchas, a DNA polymerase, a sequencing primer) immobilized or attached to asolid support such as the bottom of the sample well. The target nucleicacid molecule or the polymerase can be attached to a sample wall, suchas at the bottom of the sample well directly or through a linker. Thesample well can also contain any other reagents needed for nucleic acidsynthesis via a primer extension reaction, such as, for example suitablebuffers, co-factors, enzymes (e.g., a polymerase) anddeoxyribonucleoside polyphosphates, such as, e.g., deoxyribonucleosidetriphosphates, including deoxyadenosine triphosphate (dATP),deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP),deoxyuridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP)dNTPs, that include luminescent tags, such as fluorophores. Each classof dNTPs (e.g. adenine-containing dNTPs (e.g., dATP),cytosine-containing dNTPs (e.g., dCTP), guanine-containing dNTPs (e.g.,dGTP), uracil-containing dNTPs (e.g., dUTPs) and thymine-containingdNTPs (e.g., dTTP)) is conjugated to a distinct luminescent tag suchthat detection of light emitted from the tag indicates the identity ofthe dNTP that was incorporated into the newly synthesized nucleic acid.Emitted light from the luminescent tag can be detected and attributed toits appropriate luminescent tag (and, thus, associated dNTP) via anysuitable device and/or method, including such devices and methods fordetection described elsewhere herein. The luminescent tag may beconjugated to the dNTP at any position such that the presence of theluminescent tag does not inhibit the incorporation of the dNTP into thenewly synthesized nucleic acid strand or the activity of the polymerase.In some embodiments, the luminescent tag is conjugated to the terminalphosphate (the gamma phosphate) of the dNTP.

The single-stranded target nucleic acid template can be contacted with asequencing primer, dNTPs, polymerase and other reagents necessary fornucleic acid synthesis. In some embodiments, all appropriate dNTPs canbe contacted with the single-stranded target nucleic acid templatesimultaneously (e.g., all dNTPs are simultaneously present) such thatincorporation of dNTPs can occur continuously. In other embodiments, thedNTPs can be contacted with the single-stranded target nucleic acidtemplate sequentially, where the single-stranded target nucleic acidtemplate is contacted with each appropriate dNTP separately, withwashing steps in between contact of the single-stranded target nucleicacid template with differing dNTPs. Such a cycle of contacting thesingle-stranded target nucleic acid template with each dNTP separatelyfollowed by washing can be repeated for each successive base position ofthe single-stranded target nucleic acid template to be identified.

The sequencing primer anneals to the single-stranded target nucleic acidtemplate and the polymerase consecutively incorporates the dNTPs (orother deoxyribonucleoside polyphosphate) to the primer via thesingle-stranded target nucleic acid template. The unique luminescent tagassociated with each incorporated dNTP can be excited with theappropriate excitation light during or after incorporation of the dNTPto the primer and its emission can be subsequently detected, using, anysuitable device(s) and/or method(s), including devices and methods fordetection described elsewhere herein. Detection of a particular emissionof light can be attributed to a particular dNTP incorporated. Thesequence obtained from the collection of detected luminescent tags canthen be used to determine the sequence of the single-stranded targetnucleic acid template via sequence complementarity.

While the present disclosure makes reference to dNTPs, devices, systemsand methods provided herein may be used with various types ofnucleotides, such as ribonucleotides and deoxyribonucleotides (e.g.,deoxyribonucleoside polyphophates with at least 4, 5, 6, 7, 8, 9, or 10phosphate groups). Such ribonucleotides and deoxyribonucleotides caninclude various types of tags (or markers) and linkers.

Signals emitted upon the incorporation of nucleosides can be stored inmemory and processed at a later point in time to determine the sequenceof the target nucleic acid template. This may include comparing thesignals to a reference signals to determine the identities of theincorporated nucleosides as a function of time. Alternative or inaddition to, signal emitted upon the incorporation of nucleoside can becollected and processed in real time (i.e., upon nucleosideincorporation) to determine the sequence of the target nucleic acidtemplate in real time.

Nucleic acid sequencing of a plurality of single-stranded target nucleicacid templates may be completed where multiple sample wells areavailable, as is the case in devices described elsewhere herein. Eachsample well can be provided with a single-stranded target nucleic acidtemplate and a sequencing reaction can be completed in each sample well.Each of the sample wells may be contacted with the appropriate reagents(e.g., dNTPs, sequencing primers, polymerase, co-factors, appropriatebuffers, etc.) necessary for nucleic acid synthesis during a primerextension reaction and the sequencing reaction can proceed in eachsample well. In some embodiments, the multiple sample wells arecontacted with all appropriate dNTPs simultaneously. In otherembodiments, the multiple sample wells are contacted with eachappropriate dNTP separately and each washed in between contact withdifferent dNTPs. Incorporated dNTPs can be detected in each sample welland a sequence determined for the single-stranded target nucleic acid ineach sample well as is described above.

Embodiments directed towards single molecule nucleic acid sequencing mayuse any polymerase that is capable of synthesizing a nucleic acidcomplementary to a target nucleic acid molecule. Examples of polymerasesinclude a DNA polymerase, an RNA polymerase, a thermostable polymerase,a wild-type polymerase, a modified polymerase, E. coli DNA polymerase I,T7 DNA polymerase, bacteriophage T4 DNA polymerase φ29 (psi29) DNApolymerase, Taq polymerase, Tth polymerase, Tli polymerase, Pfupolymerase, Pwo polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taqpolymerase, LA-Taq polymerase, Sso polymerase, Poc polymerase, Pabpolymerase, Mth polymerase, ES4 polymerase, Tru polymerase, Tacpolymerase, Tne polymerase, Tma polymerase, Tca polymerase, Tihpolymerase, Tfi polymerase, Platinum Taq polymerases, Tbr polymerase,Tfl polymerase, Tth polymerase, Pfutubo polymerase, Pyrobest polymerase,Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenowfragment, polymerase with 3′ to 5′ exonuclease activity, and variants,modified products and derivatives thereof. In some embodiments, thepolymerase is a single subunit polymerase. In some embodiments, thepolymerase is a polymerase with high processivity. Polymeraseprocessivity generally refers to the capability of a polymerase toconsecutively incorporate dNTPs into a nucleic acid template withoutreleasing the nucleic acid template. Upon base pairing between anucleobase of a target nucleic acid and the complementary dNTP, thepolymerase incorporates the dNTP into the newly synthesized nucleic acidstrand by forming a phosphodiester bond between the 3′ hydroxyl end ofthe newly synthesized strand and the alpha phosphate of the dNTP. Inexamples in which the luminescent tag conjugated to the dNTP is afluorophore, its presence is signaled by excitation and a pulse ofemission is detected during or after the step of incorporation. Fordetection labels that are conjugated to the terminal (gamma) phosphateof the dNTP, incorporation of the dNTP into the newly synthesized strandresults in release of the beta and gamma phosphates and the detectionlabel, which is free to diffuse in the sample well, resulting in adecrease in emission detected from the fluorophore.

Embodiments directed toward single molecule RNA sequencing may use anyreverse transcriptase that is capable of synthesizing complementary DNA(cDNA) from an RNA template. In such embodiments, a reversetranscriptase can function in a manner similar to polymerase in thatcDNA can be synthesized from an RNA template via the incorporation ofdNTPs to a reverse transcription primer annealed to an RNA template. ThecDNA can then participate in a sequencing reaction and its sequencedetermined as described above. The determined sequence of the cDNA canthen be used, via sequence complementarity, to determine the sequence ofthe original RNA template. Examples of reverse transcriptases includeMoloney Murine Leukemia Virus reverse transcriptase (M-MLV), avianmyeloblastosis virus (AMV) reverse transcriptase, human immunodeficiencyvirus reverse transcriptase (HIV-1) and telomerase reversetranscriptase.

Having recognized the need for simple, less complex apparatuses forperforming single molecule detection and/or nucleic acid sequencing, thepresent disclosure provides techniques for detecting single moleculesusing sets of tags, such as optical (e.g., luminescent) tags, to labeldifferent molecules. Such single molecules may be nucleotides or aminoacids having tags. Tags may be detected while bound to single molecules,upon release from the single molecules, or while bound to and uponrelease from the single molecules. In some examples, tags areluminescent tags. Each luminescent tag in a selected set is associatedwith a respective molecule. For example, a set of four tags may be usedto “label” the nucleobases present in DNA—each tag of the set beingassociated with a different nucleobase, e.g., a first tag beingassociated with adenine (A), a second tag being associated with cytosine(C), a third tag being associated with guanine (G), and a fourth tagbeing associated with thymine (T). Moreover, each of the luminescenttags in the set of tags has different properties that may be used todistinguish a first tag of the set from the other tags in the set. Inthis way, each tag is uniquely identifiable using one or more of thesedistinguishing characteristics. By way of example and not limitation,the characteristics of the tags that may be used to distinguish one tagfrom another may include the emission energy and/or wavelength of thelight that is emitted by the tag in response to excitation and/or theenergy and/or wavelength of the excitation light that excites aparticular tag.

Embodiments may use any suitable combination of tag characteristics todistinguish a first tag in a set of tags from the other tags in the sameset. For example, some embodiments may use only the wavelength of theemission light from the tags to identify the tags. In such embodiments,each tag in a selected set of tags has a different peak emissionwavelength from the other tags in the set and the luminescent tags areall excited by light from a single excitation source. FIG. 1-1illustrates the emission spectra from four luminescent tags according toan embodiment where the four tags exhibit their respective intensitypeak at different emission wavelengths, referred to herein as the tag's“peak emission wavelength.” A first emission spectrum 1-101 from a firstluminescent tag has a peak emission wavelength at λ1, a second emissionspectrum 1-102 from a second luminescent tag has a peak emissionwavelength at λ2, a third emission spectrum 1-103 from a thirdluminescent tag has a peak emission wavelength at λ3, and a fourthemission spectrum 1-104 from a fourth luminescent tag has a peakemission wavelength at λ4. In this embodiment, the emission peaks of thefour luminescent tags may have any suitable values that satisfy therelation λ1<λ2<λ3<λ4. The four emission spectra may or may not overlap.However, if the emission spectra of two or more tags overlap, it isdesirable to select a luminescent tag set such that one tag emitssubstantially more light than any other tag at each respective peakwavelength. In this embodiment, the excitation wavelength at which eachof the four tags maximally absorbs light from the excitation source issubstantially the same, but that need not be the case. Using the abovetag set, four different molecules may be labeled with a respective tagfrom the tag set, the tags may be excited using a single excitationsource, and the tags can be distinguished from one another by detectingthe emission wavelength of the tags using an optical system and sensors.While FIG. 1-1 illustrates four different tags, it should be appreciatedthat any suitable number of tags may be used.

Other embodiments may use both the wavelength of the emission light fromthe tags and the wavelength at which the tags absorb excitation light toidentify the tags. In such embodiments, each tag in a selected set oftags has a different combination of emission wavelength and excitationwavelength from the other tags in the set. Thus, some tags within aselected tag set may have the same emission wavelength, but be excitedby light of different wavelengths. Conversely, some tags within aselected tag set may have the same excitation wavelength, but emit lightat different wavelengths. FIG. 1-2A illustrates the emission spectrafrom four luminescent tags according to an embodiment where two of thetags have a first peak emission wavelength and the other two tags have asecond peak emission wavelength. A first emission spectrum 1-105 from afirst luminescent tag has a peak emission wavelength at λ1, a secondemission spectrum 1-106 from a second luminescent tag also has a peakemission wavelength at λ1, a third emission spectrum 1-107 from a thirdluminescent tag has a peak emission wavelength at λ2, and a fourthemission spectrum 1-108 from a fourth luminescent tag also has a peakemission wavelength at λ2. In this embodiment, the emission peaks of thefour luminescent tags may have any suitable values that satisfy therelation λ1<λ2. FIG. 1-2B illustrates the absorption spectra from thefour luminescent tags, where two of the tags have a first peakabsorption wavelength and the other two tags have a second peakabsorption wavelength. A first absorption spectrum 1-109 for the firstluminescent tag has a peak absorption wavelength λ3, a second absorptionspectrum 1-110 for the second luminescent tag has a peak absorptionwavelength at λ4, a third absorption spectrum 1-111 for the thirdluminescent tag has a peak absorption wavelength at λ3, and a fourthabsorption spectrum 1-112 for the fourth luminescent tag has a peakabsorption wavelength at λ4. Note that the tags that share an emissionpeak wavelength in FIG. 1-2A do not share an absorption peak wavelengthin FIG. 1-2B. Using such a tag set allows distinguishing between fourtags even when there are only two emission wavelengths for the fourdyes. This is possible using two excitation sources that emit atdifferent wavelengths or a single excitation source capable of emittingat multiple wavelengths. If the wavelength of the excitation light isknown for each detected emission event, then it can be determined whichtag was present. The excitation source(s) may alternate between a firstexcitation wavelength and a second excitation wavelength, which isreferred to as interleaving. Alternatively, two or more pulses of thefirst excitation wavelength may be used followed by two or more pulsesof the second excitation wavelength.

While not illustrated in the figures, other embodiments may determinethe identity of a luminescent tag based on the absorption frequencyalone. Such embodiments are possible if the excitation light can betuned to specific wavelengths that match the absorption spectrum of thetags in a tag set. In such embodiments, the optical system and sensorused to direct and detect the light emitted from each tag does not needto be capable of detecting the wavelength of the emitted light. This maybe advantageous in some embodiments because it reduces the complexity ofthe optical system and sensors because detecting the emission wavelengthis not required in such embodiments.

As discussed above, the inventors have recognized and appreciated theneed for being able to distinguish different luminescent tags from oneanother using various characteristics of the tags. The type ofcharacteristics used to determine the identity of a tag impact thephysical device used to perform this analysis. The present applicationdiscloses several embodiments of an apparatus, device, instrument andmethods for performing these different experiments.

Briefly, the inventors have recognized and appreciated that a pixelateddevice with a large number of pixels (e.g., hundreds, thousands,millions or more) allows for the detection of a plurality of individualmolecules or particles in parallel. The molecules may be, by way ofexample and not limitation, proteins and/or DNA. Moreover, a high-speeddevice that can acquire data at more than one hundred frames per secondallows for the detection and analysis of dynamic processes or changesthat occur over time within the sample being analyzed.

The compact apparatus described herein may be used to bring automatedbioanalytics to regions of the world that previously could not performquantitative analysis of biological samples. For example, blood testsfor infants may be performed by placing a blood sample on a disposableor recyclable integrated assay chip (also referred to herein as an“integrated device”), placing the integrated device into the small,portable instrument for analysis, and processing the results by acomputer that connects to the instrument for immediate review by a user.The data may also be transmitted over a data network to a remotelocation to be analyzed, and/or archived for subsequent clinicalanalyses. Alternatively, the instrument may include one or moreprocessors capable of analyzing data obtained from the integrateddevice, and provide results for review without the need of an externalcomputer.

II. Overview of Apparatus

A schematic overview of apparatus 2-100 for analyzing specimens isillustrated in FIG. 2-1. According to some embodiments, the apparatus2-100 comprises an integrated assay chip (also referred to herein as an“integrated device”) 2-110 and a base instrument 2-120, into which theintegrated device may be inserted. The base instrument 2-120 maycomprise a computer interface 2-124, at least one electronic processor2-123, and a user interface 2-125. The integrated device may comprise aninstrument interface 2-130, at least one sample well 2-111, at least oneexcitation source 2-121, and at least one sensor 2-122, though inpreferred embodiments, there will be a plurality of sample wells,excitation sources, and sensors disposed on an integrated device 2-110.According to some embodiments, the instrument 2-120 includes anysuitable socket for interfacing with the integrated device 2-110. Forexample, the instrument 2-120 may include a socket (not illustrated)comprising mechanical registration and multi-pin electrical connectionfor receiving the integrated device 2-110.

In some embodiments, a computer interface 2-124 is used to connect witha computing device 2-130. Any suitable computer interface 2-124 andcomputing device 2-130 may be used. For example, the computer interface2-124 may be a USB interface or a Firewire interface. The computingdevice 2-130 may be any general purpose computer, such as a laptop ordesktop computer. The computer interface 2-124 facilitates communicationof information between the instrument 2-120 and the computing device2-130. Input information for controlling and/or configuring theinstrument 2-120 may be provided through the computing device 2-130connected to the computer interface 2-124 of the instrument.Additionally, output information from the instrument may be received bythe computing device 2-130 through the computer interface 2-124. Suchoutput information may include feedback about performance of theinstrument 2-120 and information relating to signals from the sensors2-122, which may comprise raw and/or processed data in some embodiments.

The instrument 2-120 may also include at least one processing device2-123 for analyzing data received from the sensors 2-122. In someembodiments, the processing device 2-123 may comprise aspecially-adapted processor (e.g., a central processing unit (CPU) suchas one or more microprocessor or microcontroller cores, afield-programmable gate array (FPGA), an application-specific integratedcircuit (ASIC), a custom integrated circuit, a digital signal processor(DSP), or a combination thereof.) Memory (not) shown may storemachine-readable instructions that specially adapt the processor 2-123to execute instrument management functions, signal collection andprocessing functions, as well as issue control signals to the integrateddevice for various purposes such as operation of the excitation sources.In some embodiments, the processing of data from the sensors 2-122 maybe performed by both the processing device 2-123 and the externalcomputing device 2-130. In other embodiments, the computing device 2-130may be omitted and processing of data from the sensor 2-122 may beperformed solely by processing device 2-123.

In some embodiments, the instrument 2-120 includes a user interface2-125 for interactive operation of the instrument. The user interface2-125 may be configured to allow a user to input information into theinstrument, such as commands and/or settings used to control thefunctioning of the instrument. In some embodiments, the user interface2-125 may include any one of or a combination of buttons, switches,dials, touch screen, touch pad, display, and microphone for receivingvoice commands Additionally, the user interface 2-125 may allow a userto receive feedback on the performance of the instrument and/orintegrated device, such as proper alignment and/or information obtainedby readout signals from the sensors on the integrated device. In someembodiments, the user interface 2-125 may provide feedback using aspeaker to provide audible feedback, and/or indicator lights and/or adisplay screen for providing visual feedback.

In some embodiments, the integrated device 2-110 comprises a pluralityof pixels, each pixel associated with its own individual sample well2-111 and its own associated sensor 2-122. The plurality of pixels maybe arranged in an array, and there may be any suitable number of pixels.For example, the integrated device may include between 100 and 1,000pixels according to some embodiments, between 1,000 and 10,000 pixelsaccording to some embodiments, between 10,000 and 100,000 pixelsaccording to some embodiments, between 100,000 and 1,000,000 pixelsaccording to some embodiments, and yet between 1,000,000 and 10,000,000pixels according to some embodiments. In some implementations, there maybe fewer or more pixels on an integrated device. The integrated device2-110 and instrument 2-120 may include multi-channel, high-speedcommunication links for handling data associated with large pixel arrays(e.g., more than 1000 pixels).

An integrated device may appear as depicted in FIG. 2-2. Electronic,optical, and related structures may all be incorporated onto a singlesubstrate 2-200. The integrated device may include an array ofactive-source pixels 2-205 and integrated electronic circuitry. Theintegrated electronic circuitry may include drive and read-out circuitry2-215 coupled to the sensors of the pixel array, and signal processingcircuitry. The signal processing circuitry may include analog-to-digitalconverters 2-217 and one or more field-programmable gate arrays and/ordigital signal processors 2-219. Some embodiments may have more circuitcomponents, and some embodiments may have fewer circuit componentsintegrated on the substrate. Although the components of the integrateddevice 2-110 are depicted on a single level in FIG. 2-2, the componentsmay be fabricated on multiple levels on the substrate 2-200.

According to some embodiments, there may be a walled chamber formedaround a plurality of pixels 2-205 on the integrated device. The walledchamber may be configured to hold a fluid specimen over the plurality ofpixels. In some implementations, there may be a cover that can closeover the walled chamber to exclude light external to the walled chamberfrom illuminating the plurality of pixels. According to someimplementations, there may be a ridge running around the plurality ofpixels 2-205, or the plurality of pixels may be formed in a depression.The walled chamber, ridge, or depression may be configured to retain afluid specimen over the plurality of pixels. The integrated device maybe inserted into a receiving dock of an instrument 2-120, and a coverclosed over the receiving dock to exclude light external to thereceiving dock from illuminating the plurality of pixels. In someembodiments, the integrated device 2-110 and chamber are packaged in asingle module. The module may have exterior electrical contacts that arearranged to electrically contact pins of a receiving dock of aninstrument 2-120.

In some embodiments, there may be optical elements (not shown) locatedon the integrated device 2-110 that are arranged for guiding andcoupling excitation energy from one or more excitation sources 2-121 tothe sample wells 2-111. Such source-to-well elements may includeplasmonic structures and other microfabricated structures locatedadjacent the sample wells. Additionally, in some embodiments, there maybe optical elements located on the integrated device that are configuredfor guiding emission energy from the sample wells 2-111 to correspondingsensors 2-122. Such well-to-sample elements may include may includeplasmonic structures and other microfabricated structures locatedadjacent the sample wells. In some embodiments, a single component mayplay a role in both in coupling excitation energy to a sample well anddelivering emission energy from the sample well to a correspondingsensor.

In some implementations, an integrated device 2-110 may include morethan one type of excitation source that is used to excite samples at asample well. For example, there may be multiple excitation sourcesconfigured to produce multiple excitation energies or wavelengths forexciting a sample. In some embodiments, a single excitation source maybe configured to emit multiple wavelengths that are used to excitesamples in the sample wells. In some embodiments, each sensor at a pixelof the integrated device 2-110 may include multiple sub-sensorsconfigured to detect different emission energy characteristics from thesample.

In operation, parallel analyses of samples within the sample wells 2-111are carried out by exciting the samples within the wells using theexcitation source 2-121 and detecting signals from sample emission withthe sensors 2-122. Emission energy from a sample may be detected by acorresponding sensor 2-122 and converted to at least one electricalsignal. The resulting signal, or signals, may be processed on theintegrated device 2-110 in some embodiments, or transmitted to theinstrument 2-120 for processing by the processing device 2-123 and/orcomputing device 2-130. Signals from a sample well may be received andprocessed independently from signals associated with the other pixels.

When an excitation source 2-121 delivers excitation energy to a samplewell, at least one sample within the well may luminesce, and theresulting emission may be detected by a sensor. As used herein, thephrases “a sample may luminesce” or “a sample may emit radiation” or“emission from a sample” mean that a luminescent tag, marker, orreporter, the sample itself, or a reaction product associated with thesample may produce the emitted radiation.

In some embodiments, samples may be labeled with one or more tags, andemission associated with the tags is discernable by the instrument. Forexample, components of the integrated device may affect the emissionfrom a sample well to produce a spatial emission distribution patternthat is dependent on the emission wavelength. A corresponding sensor forthe sample well may be configured to detect the spatial distributionpatterns from a sample well and produce signals that differentiatebetween the different emission wavelengths, as described in furtherdetail below.

Various tags, markers, or reporters may be used with the integrateddevice and instrument. Luminescent markers (also referred to herein as“markers”) may be exogenous or endogenous markers. Exogenous markers maybe external luminescent markers used as a reporter and/or tag forluminescent labeling. Examples of exogenous markers may include but arenot limited to fluorescent molecules, fluorophores, fluorescent dyes,fluorescent stains, organic dyes, fluorescent proteins, species thatparticipate in fluorescence resonance energy transfer (FRET), enzymes,and/or quantum dots. Other exogenous markers are known in the art. Suchexogenous markers may be conjugated to a probe or functional group(e.g., molecule, ion, and/or ligand) that specifically binds to aparticular target or component. Attaching an exogenous tag or reporterto a probe allows identification of the target through detection of thepresence of the exogenous tag or reporter. Examples of probes mayinclude proteins, nucleic acid (e.g., DNA, RNA) molecules, lipids andantibody probes. The combination of an exogenous marker and a functionalgroup may form any suitable probes, tags, and/or labels used fordetection, including molecular probes, labeled probes, hybridizationprobes, antibody probes, protein probes (e.g., biotin-binding probes),enzyme labels, fluorescent probes, fluorescent tags, and/or enzymereporters.

Although the present disclosure makes reference to luminescent markers,other types of markers may be used with devices, systems and methodsprovided herein. Such markers may be mass tags, electrostatic tags, orelectrochemical labels.

While exogenous markers may be added to a sample, endogenous markers maybe already part of the sample. Endogenous markers may include anyluminescent marker present that may luminesce or “autofluoresce” in thepresence of excitation energy. Autofluorescence of endogenousfluorophores may provide for label-free and noninvasive labeling withoutrequiring the introduction of exogenous fluorophores. Examples of suchendogenous fluorophores may include hemoglobin, oxyhemoglobin, lipids,collagen and elastin crosslinks, reduced nicotinamide adeninedinucleotide (NADH), oxidized flavins (FAD and FMN), lipofuscin,keratin, and/or prophyrins, by way of example and not limitation.

FIG. 2-3 depicts components of a computing device 2-310. Some or all ofthe components shown may be present in embodiments of an apparatus 2-100for analyzing specimens. In a distributed computing environment, somecomponents may be located on a server and some components may be locatedon a client device. In some implementations, a computing device 2-130 incommunication with a base instrument 2-120 may comprise some or allcomponents of a computing system 2-300 that is depicted in FIG. 2-3. Insome embodiments, a base instrument 2-120 may include some or all of thecomponents of a computing device 2-310.

Components of computing device 2-310 may include, but are not limitedto, a processing unit 2-320, a memory 2-330, and a bus 2-321 thatcouples various components including the memory to the processing unit2-320. The bus 2-321 may be any of several types of bus structuresincluding a memory bus or memory controller, a peripheral bus, and alocal bus using any of a variety of bus architectures. By way ofexample, and not limitation, such architectures include IndustryStandard Architecture (ISA) bus, Micro Channel Architecture (MCA) bus,Enhanced ISA (EISA) bus, Video Electronics Standards Association (VESA)local bus, and Peripheral Component Interconnect (PCI) bus also known asMezzanine bus.

Computer 2-310 may include one or more types of machine-readable media.Machine-readable media can be any available media that can be accessedby computer 2-310 and includes both volatile and nonvolatile,manufactured storage media, removable and non-removable manufacturedstorage media. By way of example, and not limitation, machine-readablemedia may comprise information such as computer-readable instructions,data structures, program modules or other data. Machine-readable mediaincludes, but is not limited to, RAM, ROM, EEPROM, flash memory or othermemory-device technology, CD-ROM, digital versatile disks (DVD) or otheroptical disk storage, magnetic cassettes, magnetic tape, magnetic diskstorage or other magnetic storage devices, or any other manufactureddata-storage device which can be used to store the desired informationand which can accessed by computer 2-310.

The memory 2-330 may include computer storage media in the form ofvolatile and/or nonvolatile memory such as read only memory (ROM) 2-331and random access memory (RAM) 2-332. A basic input/output system 2-333(BIOS), containing the basic routines that help to transfer informationbetween elements within computer 2-310, such as during start-up, may bestored in ROM 2-331. RAM 2-332 may contain data and/or program modulesthat are immediately accessible to and/or presently being operated on byprocessing unit 2-320. By way of example, and not limitation, FIG. 2-3illustrates an operating system 2-334, application programs 2-335, otherprogram modules 2-336, and program data 2-337.

The computer 2-310 may also include other removable/non-removable,volatile/nonvolatile machine-readable media. By way of example only,FIG. 2-3 illustrates a hard disk drive 2-341 that reads from or writesto non-removable, nonvolatile magnetic media, a magnetic disk drive2-351 that reads from or writes to a removable, nonvolatile magneticdisk 2-352, and an optical disk drive 2-355 that reads from or writes toa removable, nonvolatile optical disk 2-356 such as a CD ROM or otheroptical media. Other removable/non-removable, volatile/nonvolatilemachine-readable media that can be used in the exemplary operatingenvironment include, but are not limited to, magnetic tape cassettes,flash memory cards, digital versatile disks, digital video tape, solidstate RAM, solid state ROM, and the like. The hard disk drive 2-341 maybe connected to the system bus 2-321 through a non-removable memoryinterface such as interface 2-340, and magnetic disk drive 2-351 andoptical disk drive 2-355 may be connected to the system bus 2-321 by aremovable memory interface, such as interface 2-350.

The drives and their associated machine-readable media discussed aboveand illustrated in FIG. 2-3, provide storage of machine-readableinstructions, data structures, program modules and other data for thecomputer 2-310. In FIG. 2-3, for example, hard disk drive 2-341 isillustrated as storing operating system 2-344, application programs2-345, other program modules 2-346, and program data 2-347. Thesecomponents may either be the same as, or different from, operatingsystem 2-334, application programs 2-335, other program modules 2-336,and program data 2-337. Operating system 2-344, application programs2-345, other program modules 2-346, and program data 2-347 are givendifferent numbers here to illustrate that, at a minimum, they aredifferent copies.

A user may enter commands and information into the computer 2-310through input devices such as a keyboard 2-362 and pointing device2-361, commonly referred to as a mouse, trackball or touch pad. Otherinput devices (not shown) may include a microphone, joystick, game pad,satellite dish, scanner, or the like. These and other input devices maybe connected to the processing unit 2-320 through a user input interface2-360 that is coupled to the system bus, but may be connected by otherinterface and bus structures, such as a parallel port, game port or auniversal serial bus (USB). A monitor 2-391 or other type of displaydevice may also be connected to the system bus 2-321 via an interface,such as a video interface 2-390. In addition to the monitor, a computingdevice 2-310 may also include other peripheral output devices such asspeakers 2-397 and printer 2-396, which may be connected through aoutput peripheral interface 2-395.

The computer 2-310 may operate in a networked environment using logicalconnections to one or more remote devices, such as a remote computer2-380. The remote computer 2-380 may be a personal computer, a server, arouter, a network PC, a peer device or other common network node, andmay include many or all of the elements described above relative to thecomputer 2-310, although only a memory storage device 2-381 has beenillustrated in FIG. 2-3. The logical connections depicted in FIG. 2-3include a local area network (LAN) 2-371 and a wide area network (WAN)2-373, but may also include other networks. Such networking environmentsmay be commonplace in offices, enterprise-wide computer networks,intranets and the Internet. Network connections may be wired, opticalfiber based, or wireless.

When used in a LAN networking environment, the computer 2-310 may beconnected to the LAN 2-371 through a network interface or adapter 2-370.When used in a WAN networking environment, the computer 2-310 mayinclude a modem 2-372 or other means for establishing communicationsover the WAN 2-373, such as the Internet. The modem 2-372, which may beinternal or external, may be connected to the system bus 2-321 via theuser input interface 2-360, or other appropriate mechanism. In anetworked environment, program modules depicted relative to the computer2-310, or portions thereof, may be stored in a remote memory storagedevice. By way of example, and not limitation, FIG. 2-3 illustratesremote application programs 2-385 as residing on memory device 2-381. Itwill be appreciated that the network connections shown are exemplary andother means of establishing a communications link between the computersmay be used.

III. Active Source Pixel and Sample Well

III. A. Active Source Pixel

Referring now to FIG. 3-1, in various embodiments, an integrated devicemay include a plurality of active source pixels 3-100. An active sourcepixel (also referred to as “pixel” herein) may comprise an excitationsource located at the pixel. The plurality of pixels may be arranged ona substrate 3-105 in a regular array (e.g., a one-dimensional ortwo-dimensional array). The pixels 3-100 may be arranged to have atleast one periodic spacing between pixels, according to one embodiment.For example, pixels along a first direction (a row direction) may have afirst periodic spacing, and pixels along a second direction (a columndirection) may have a second periodic spacing. However, someimplementations may not have regular periodic spacings between pixels,or may have other arrangements of the pixels that may include more thantwo periodic spacings. There may be between 100 pixels and 1 millionpixels on an integrated device 2-110, though in some embodiments, theremay be more pixels on an integrated device.

As depicted in FIG. 3-1 and according to one embodiment of an integrateddevice 2-110, an active pixel may include a sample well 3-210 in whichat least one sample 3-101 from a specimen may be retained forobservation. A pixel may further include at least one excitation source3-240 that provides energy to excite a sample in the sample well and asensor 3-260 that detects emission from the sample. According to someembodiments, a pixel 3-100 may include additional structures. Forexample, a pixel 3-100 may include an excitation-coupling structure3-220 that affects coupling of excitation energy to a sample within thesample well. A pixel may also include an emission-coupling structure3-250 that affects coupling of emission energy from a sample within thewell to the sensor 3-260.

In some embodiments, a pixel 3-100 may include at least one integratedcomplementary metal-oxide-semiconductor (CMOS) device (e.g., at leastone integrated amplifier, at least one gating transistor, etc, not shownin the drawing) that is used for processing signals from the sensor3-260. Integrated CMOS circuitry may be located on one or more levelsnear the sensor 3-260. Ground planes and/or interconnects may also belocated within a pixel of an integrated device.

The arrangement of components in a pixel is not limited to those shownin FIG. 3-1. In some embodiments, the first structure 3-220, excitationsource 3-240, second structure 3-250, and sensor 3-260 may be arrangedin an order, from top to bottom, different than shown in the drawing.

III. B Sample Well Embodiments

According to some embodiments, a sample well 3-210 may be formed at oneor more pixels of an integrated device. A sample well may comprise asmall volume or region formed at a surface of a substrate 3-105 andarranged such that samples 3-101 may diffuse into and out of the samplewell from a specimen deposited on the surface of the substrate, asdepicted in FIG. 3-1 and FIG. 3-2. The sample well 3-210 may have alength or depth extending in a direction normal to the substratesurface, sometimes referred to as a longitudinal direction of the samplewell. In various embodiments, a sample well 3-210 may be arranged toreceive excitation energy from an excitation source 3-240. Samples 3-101that diffuse into the sample well may be retained, temporarily orpermanently, within an excitation region 3-215 of the sample well by anadherent 3-211. In the excitation region, a sample may be excited byexcitation energy (e.g., excitation radiation 3-247), and subsequentlyemit radiation that may be observed and evaluated to characterize thesample.

In further detail of operation, at least one sample 3-101 to be analyzedmay be introduced into a sample well 3-210, e.g., from a specimen (notshown) containing a fluid suspension of samples. Energy from anexcitation source 3-240 on the substrate may excite the sample or atleast one tag (also referred to as a biological marker, reporter, orprobe) attached to the sample or otherwise associated with the samplewhile it is within an excitation region 3-215 within the sample well.According to some embodiments, a tag may be a luminescent molecule(e.g., a luminescent tag or probe) or quantum dot. In someimplementations, there may be more than one tag that is used to analyzea sample (e.g., distinct tags that are used for single-molecule geneticsequencing as described in “Real-Time DNA Sequencing from SinglePolymerase Molecules,” by J. Eid, et al., Science 323, p. 133 (2009),which is incorporated by reference). During and/or after excitation, thesample or tag may emit emission energy. When multiple tags are used,they may emit at different characteristic energies and/or emit withdifferent temporal characteristics. The emissions from the sample wellmay radiate or otherwise travel to a sensor 3-260 where they aredetected and converted into electrical signals that can be used tocharacterize the sample.

According to some embodiments, a sample well 3-210 may be a partiallyenclosed structure, as depicted in FIG. 3-2. In some implementations, asample well 3-210 comprises a sub-micron-sized hole or opening(characterized by at least one transverse dimension D_(sw)) formed in atleast one layer of material 3-230. In some cases, the hole may bereferred to as a “nanohole.” The transverse dimension of the sample wellmay be between approximately 20 nanometers and approximately 1 micron,according to some embodiments, though larger and smaller sizes may beused in some implementations. A volume of the sample well 3-210 may bebetween about 10⁻²¹ liters and about 10⁻¹⁵ liters, in someimplementations. A sample well may be formed as a waveguide that may, ormay not, support a propagating mode. In some embodiments, a sample wellmay be formed as a zero-mode waveguide (ZMW) having a cylindrical shape(or similar shape) with a diameter (or largest transverse dimension)D_(sw). A ZMW may be formed in a single metal layer as a nanoscale holethat does not support a propagating optical mode through the hole.

Because the sample well 3-210 has a small volume, detection ofsingle-sample events (e.g., single-molecule events) at each pixel may bepossible even though samples may be concentrated in an examined specimenat concentrations that are similar to those found in naturalenvironments. For example, micromolar concentrations of the sample maybe present in a specimen that is placed in contact with the integrateddevice, but at the pixel level only about one sample (or single moleculeevent) may be within a sample well at any given time. Statistically,some sample wells may contain no samples and some may contain more thanone sample. However, an appreciable number of sample wells may contain asingle sample (e.g. at least 30% in some embodiments), so thatsingle-molecule analysis can be carried out in parallel for a largenumber of pixels. Because single-molecule or single-sample events may beanalyzed at each pixel, the integrated device makes it possible todetect rare events that may otherwise go unnoticed in ensemble averages.

A transverse dimension D_(sw) of a sample well may be between about 500nanometers (nm) and about one micron in some embodiments, between about250 nm and about 500 nm in some embodiments, between about 100 nm andabout 250 nm in some embodiments, and yet between about 20 nm and about100 nm in some embodiments. According to some implementations, atransverse dimension of a sample well is between approximately 80 nm andapproximately 180 nm, or between approximately one-quarter andone-eighth of the excitation wavelength or emission wavelength. In someembodiments, the depth or height of the sample well 3-210 may be betweenabout 50 nm and about 500 nm. In some implementations, the depth orheight of the sample well 3-210 may be between about 80 nm and about 250nm.

A sample well 3-210 having a sub-wavelength, transverse dimension canimprove operation of a pixel 3-100 of an integrated device 2-110 in atleast two ways. For example, excitation energy incident on the samplewell from a side opposite the specimen may couple into the excitationregion 3-215 with an exponential decay in power, and not propagate as apropagating mode through the sample well to the specimen. As a result,excitation energy is increased in the excitation region where it excitesa sample of interest, and is reduced in the specimen where it may exciteother samples that may contribute to background noise. Also, emissionfrom a sample retained at a base of the well (e.g., nearer to the sensor3-260) is preferably directed toward the sensor, since emissionpropagating up through the sample well is highly suppressed. Both ofthese effects can improve signal-to-noise ratio at the pixel. Theinventors have recognized several aspects of the sample well that can beimproved to further boost signal-to-noise levels at the pixel. Theseaspects relate to well shape and structure, and also to adjacent opticaland plasmonic structures (described below) that aid in couplingexcitation energy to the sample well and emitted radiation from thesample well.

According to some embodiments, a sample well 3-210 may be formed as asub-cutoff nanoaperture (SCN). For example, the sample well 3-210 maycomprise a cylindrically-shaped hole or bore in a conductive layer. Thecross-section of a sample well need not be round, and may be elliptical,square, rectangular, or polygonal in some embodiments. Excitation energy3-247 (e.g., optical radiation) may enter the sample well through anentrance aperture 3-212 that may be defined by walls 3-214 of the samplewell at a first end of the well, as depicted in FIG. 3-2. When formed asan SCN, the excitation energy may decay exponentially along a length ofthe SCN (e.g., in the direction of the specimen). In someimplementations, the waveguide may comprise an SCN for emitted radiationfrom the sample, but may not be an SCN for excitation energy. Forexample, the aperture and waveguide formed by the sample well may belarge enough to support a propagating mode for the excitation energy,since it may have a shorter wavelength than the emitted radiation. Theemission, at a longer wavelength, may be beyond a cut-off wavelength fora propagating mode in the waveguide. According to some embodiments, thesample well 3-210 may comprise an SCN for the excitation energy, suchthat the greatest intensity of excitation energy is localized to anexcitation region 3-215 of the sample well at an entrance to the samplewell 3-210 (e.g., localized near the interface between layer 3-235 andlayer 3-230 as depicted in the drawing). Such localization of theexcitation energy can improve localization of emission energy from thesample, and limit the observed emission to that emitted from a singlesample (e.g., a single molecule).

An example of excitation localization near an entrance of a sample wellthat comprises an SCN is depicted in FIG. 3-3. A numerical simulationwas carried out to determine intensity of excitation radiation withinand near a sample well 3-210 formed as an SCN. The results show that theintensity of the excitation radiation is about 70% of the incidentenergy at an entrance aperture of the sample well and drops to about 20%of the incident intensity within about 100 nm in the sample well. Forthis simulation, the characteristic wavelength of the excitation energywas 633 nm and the diameter of the sample well 3-210 was 140 nm. Thesample well 3-210 was formed in a layer of gold metal. Each horizontaldivision in the graph is 50 nm. As shown by the graph, more thanone-half of the excitation energy received in the sample well islocalized to about 50 nm within the entrance aperture 3-212 of thesample well.

To improve the intensity of excitation energy that is localized at thesample well, other sample well structures were developed and studied bythe inventors. FIG. 3-4 depicts an embodiment of a sample well thatincludes a cavity or divot 3-216 at an excitation end of the sample well3-210. As can be seen in the simulation results of FIG. 3-3, a region ofhigher excitation intensity exists just before the entrance aperture3-212 of the sample well. Adding a divot 3-216 to extend the samplewell, as depicted in FIG. 3-4 for example, allows a sample to move intoa region of higher excitation intensity, according to some embodiments.In some implementations, the shape and structure of the divot alters thelocal excitation field (e.g., because of a difference in refractiveindex between the layer 3-235 and fluid in the sample well), and canfurther increase the intensity of the excitation energy in the divot.

The divot may have any suitable shape. The divot may have a transverseshape that is substantially equivalent to a transverse shape of thesample well, e.g., round, elliptical, square, rectangular, polygonal,etc. In some embodiments, the sidewalls of the divot may besubstantially straight and vertical, like the walls of the sample well.In some implementations, the sidewalls of the divot may be sloped and/orcurved, as depicted in the drawing. The transverse dimension of thedivot may be approximately the same size as the transverse dimension ofthe sample well in some embodiments, may be smaller than the transversedimension of the sample well in some embodiments, or may be larger thanthe transverse dimension of the sample well in some embodiments. Thedivot 3-216 may extend between approximately 10 nm and approximately 200nm beyond the sample well. In some implementations, the divot may extendbetween approximately 50 nm and approximately 150 nm beyond the samplewell. By forming the divot, the excitation region 3-215 may extendoutside the region of the sample well that is surrounded by the layer3-230, as depicted in FIG. 3-4.

FIG. 3-5 depicts improvement of excitation energy at the excitationregion for a sample well containing a divot (shown in the leftsimulation image). For comparison, the excitation field is alsosimulated for a sample well without a divot, shown on the right. Thefield magnitude has been converted from a color rendering in theseplots, and the dark region at the base of the divot represents higherintensity than the light region within the sample well. The dark regionsabove the sample well represents the lowest intensity. As can be seen,the divot allows a sample 3-101 to move to a region of higher excitationintensity, and the divot also increases the localization of region ofhighest intensity at an excitation end of the sample well. Note that theregion of high intensity is more distributed for the sample well withoutthe divot. In some embodiments, the divot 3-216 provides an increase inexcitation energy at the excitation region by a factor of two or more.In some implementations, an increase of more than a factor of two can beobtained depending on the shape and length of the divot. In thesesimulations, the sample well comprises a layer 3-230 of Al that is 100nm thick, with a divot 3-216 that is 50 nm deep, with excitation energyat 635 nm wavelength.

FIG. 3-6 depicts another embodiment of a sample well 3-210 in which thesample well and divot are formed using a protrusion 3-615 at a surfaceof a substrate. A resulting structure for the sample well may increasethe excitation energy at the sample by more than a factor of twocompared to a sample well shown in FIG. 3-1, and may condense emissionfrom the sample well to a sensor 3-260. According to some embodiments, aprotrusion 3-615 is patterned in a first layer 3-610 of material. Theprotrusion may be formed as a circular pedestal in some implementations,and a second layer 3-620 of material may be deposited over the firstlayer and the protrusion. At the protrusion, the second layer may form ashape above the protrusion that approximates a spherical portion 3-625,as depicted. In some embodiments, a conductive layer 3-230 (e.g., areflective metal) may be deposited over the second layer 3-620 andpatterned to form a sample well 3-210 in the conductive layer above theprotrusion. A divot 3-216 may then be etched into the second layer. Thedivot may extend between about 50 nm and about 150 nm below theconductive layer 3-230. According to some embodiments, the first layer3-610 and second layer 3-620 may be optically transparent, and may ormay not be formed of a same material. In some implementations, the firstlayer 3-610 may be formed from an oxide (e.g., SiO₂) or a nitride (e.g.,Si₃N₄), and the second layer 3-620 may be formed from an oxide or anitride.

According to some embodiments, the conductive layer 3-230 above theprotrusion 3-625 is shaped approximately as a spherical reflector 3-630.The shape of the spherical portion may be controlled by selection of theprotrusion height h, diameter or transverse dimension w of theprotrusion, and a thickness t of the second layer 3-620. The location ofthe excitation region and position of the sample can be adjusted withrespect to an optical focal point of the spherical reflector byselection of the divot depth d. It may be appreciated that the sphericalreflector 3-630 can concentrate excitation energy at the excitationregion 3-215, and can also collect radiation emitted from a sample andreflect and concentrate the radiation toward the sensor 3-260.

As noted above, a sample well may be formed in any suitable shape, andis not limited to only cylindrical shapes. In some implementations, asample well may be conic, tetrahedron, pentahedron, etc. FIG. 3-7A-FIG.3-7F illustrates some example sample well shapes and structures that maybe used in some embodiments. A sample well 3-210 may be formed to havean entrance aperture 3-212 that is larger than an exit aperture 3-218for the excitation energy, according to some embodiments. The sidewallsof the sample well may be tapered or curved. Forming a sample well inthis manner can admit more excitation energy to the excitation region,yet still appreciably attenuate excitation energy that travels towardthe specimen. Additionally, emission radiated by a sample maypreferentially radiate toward the end of the sample well with the largeraperture, because of favorable energy transfer in that direction.

In some embodiments, a divot 3-216 may have a smaller transversedimension than the base of the sample well, as depicted in FIG. 3-7B. Asmaller divot may be formed by coating sidewalls of the sample well witha sacrificial layer before etching the divot, and subsequently removingthe sacrificial layer. A smaller divot may be formed to retain a samplein a region that is more equidistant from the conductive walls of thesample well. Retaining a sample equidistant from the walls of the samplewell may reduce undesirable effects of the sample well walls on theradiating sample, e.g., quenching of emission, and/or altering ofradiation lifetimes.

FIG. 3-7C and 3-7D depict another embodiment of a sample well. Accordingto this embodiment, a sample well 3-210 may compriseexcitation-energy-enhancing structures 3-711 and an adherent 3-211formed adjacent the excitation-energy-enhancing structures. Theenergy-enhancing structures 3-711 may comprise surface plasmon ornano-antenna structures formed in conductive materials on an opticallytransparent layer 3-235, according to some embodiments. FIG. 3-7Cdepicts an elevation view of the sample well 3-210 and nearby structure,and FIG. 3-7D depicts a plan view. The excitation-energy-enhancingstructures 3-711 may be shaped and arranged to enhance excitation energyin a small localized region. For example, the structures may includepointed conductors having acute angles at the sample well that increasethe intensity of the excitation energy within an excitation region3-215. In the depicted example, the excitation-energy-enhancingstructures 3-711 are in the form of a bow-tie. Samples 3-101 diffusinginto the region may be retained, temporarily or permanently, by theadherent 3-211 and excited by excitation energy that may be deliveredfrom an excitation source 3-240 located adjacent the sample well 3-210.According to some embodiments, the excitation energy may drivesurface-plasmon waves in the energy-enhancing structures 3-711. Theresulting surface-plasmon waves may produce high electric fields at thesharp points of the structures 3-711, and these high fields may excite asample retained in the excitation region 3-215. In some embodiments, asample well 3-210 depicted in FIG. 3-7C may include a divot 3-216.

Another embodiment of a sample well is depicted in FIG. 3-7E, and showsan excitation-energy-enhancing structure 3-720 formed along interiorwalls of the sample well 3-210. The excitation-energy-enhancingstructure 3-720 may comprise a metal or conductor, and may be formedusing an angled (or shadow), directional deposition where the substrateon which the sample well is formed is rotated during the deposition.During the deposition, the base of the sample well 3-210 is obscured bythe upper walls of the well, so that the deposited material does notaccumulate at the base. The resulting structure 3-720 may form an acuteangle 3-722 at the bottom of the structure, and this acute angle of theconductor can enhance excitation energy within the sample well.

In an embodiment as depicted in FIG. 3-7E, the material 3-232 in whichthe sample well is formed need not be a conductor, and may be anysuitable dielectric. According to some implementations, the sample well3-210 and excitation-energy-enhancing structure 3-720 may be formed at ablind hole etched into a dielectric layer 3-235, and a separate layer3-232 need not be deposited.

In some implementations, a shadow evaporation may be subsequentlyperformed on the structure shown in FIG. 3-7E to deposit a metallic orconductive energy-enhancing structure, e.g., a trapezoidal structure orpointed cone at the base of the sample well, as depicted by the dashedline. The energy-enhancing structure may enhance the excitation energywithin the well via surface plasmons. After the shadow evaporation, aplanarizing process (e.g., a chemical-mechanical polishing step or aplasma etching process) may be performed to remove or etch back thedeposited material at the top of the sample well, while leaving theenergy-enhancing structure within the well.

In some embodiments, a sample well 3-210 may be formed from more than asingle metal layer. FIG. 3-7F illustrates a sample well formed in amulti-layer structure, where different materials may be used for thedifferent layers. According to some embodiments, a sample well 3-210 maybe formed in a first layer 3-232 (which may be a semiconducting orconducting material), a second layer 3-234 (which may be an insulator ordielectric), and a third layer 3-230 (which may be a conductor orsemiconductor). In some embodiments, a degeneratively-dopedsemiconductor or graphene may be used for a layer of the sample well. Insome implementations, a sample well may be formed in two layers, and inother implementations a sample well may be formed in four or morelayers. In some embodiments, multi-layer materials used for forming asample well may be selected to increase surface-plasmon generation at abase of the sample well or suppress surface-plasmon radiation at a topof the well. In some embodiments, multi-layer materials used for forminga sample well may be selected to suppress excitation radiation frompropagating beyond the sample well and multi-layer structure into thebulk specimen.

In some embodiments, multi-layer materials used for forming a samplewell may be selected to increase or suppress interfacial excitons whichmay be generated by excitation radiation incident on the sample well.For example, multi-excitons, such as biexcitons and triexitons, may begenerated at an interface between two different semiconductor layersadjacent a sample well. The sample well may be formed in both the metallayer and the first semiconductor layer such that the interface betweenthe first semiconductor layer and a second semiconductor layer is at anexcitation region 3-215 of the sample well. Interfacial excitons mayhave longer lifetimes than excitons within the volume of a singlesemiconductor layer, increasing the likelihood that the excitons willexcite a sample or tag via FRET or DET. In some embodiments, at leastone quantum dot at which multi-excitons may be excited may be attachedto a bottom of the sample well (e.g., by a linking molecule). Excitonsexcited at a quantum dot may also have longer lifetimes than excitonswithin the volume of a single semiconductor layer. Interfacial excitonsor excitons generated at a quantum dot may increase the rate of FRET orDET, according to some embodiments.

Various materials may be used to form sample wells described in theforegoing embodiments. According to some embodiments, a sample well3-210 may be formed from at least one layer of material 3-230, which maycomprise any one of or a combination of a conductive material, asemiconductor, and an insulator. In some embodiments, the sample well3-210 comprises a highly conductive metallic layer, e.g., gold, silver,aluminum, copper. In some embodiments, the layer 3-230 may comprise amulti-layer stack that includes any one of or a combination of gold,silver, aluminum, copper, titanium, titanium nitride, palladium,platinum, and chromium. In some implementations, other metals may beused additionally or alternatively. According to some embodiments, asample well may comprise an alloy such as AlCu or AlSi.

In some embodiments, the multiple layers of different metals or alloysmay be used to form a sample well. In some implementations, the materialin which the sample well 3-210 is formed may comprise alternating layersof metals and non-metals, e.g., alternating layers of metal and one ormore oxides. In some embodiments, the non-metal may include a polymer,such as polyvinyl phosphonic acid or a polyethylene glycol (PEG)-thiol.

A layer 3-230 in which a sample well is formed may be deposited on oradjacent to at least one optically transparent layer 3-235, according tosome embodiments, so that excitation energy (in the form of optical) andemission energy (in the form of optical) may travel to and from thesample well 3-210 without significant attenuation. For example,excitation energy from an excitation source 3-240 may pass through theat least one optically transparent layer 3-235 to the excitation region3-215, and emission from the sample may pass through the same layer orlayers to the sensor 3-260.

In some embodiments, at least one surface of the sample well 3-210 maybe coated with one or more layers 3-211, 3-280 of material that affectthe action of a sample within the sample well, as depicted in FIG. 3-8.For example, a thin dielectric layer 3-280 (e.g., alumina, titaniumnitride, or silica) may be deposited as a passivating coating onsidewalls of the sample well. Such a coating may be implemented toreduce sample adhesion of a sample outside the excitation region 3-215,or to reduce interaction between a sample and the material 3-230 inwhich the sample well 3-210 is formed. The thickness of a passivatingcoating within the sample well may be between about 5 nm and about 50nm, according to some embodiments.

In some implementations, a material for a coating layer 3-280 may beselected based upon an affinity of a chemical agent for the material, sothat the layer 3-280 may be treated with a chemical or biologicalsubstance to further inhibit adhesion of a sample species to the layer.For example, a coating layer 3-280 may comprise alumina, which may bepassivated with a polyphosphonate passivation layer, according to someembodiments. Additional or alternative coatings and passivating agentsmay be used in some embodiments.

According to some embodiments, at least a bottom surface of the samplewell 3-210 and/or divot 3-216 may be treated with a chemical orbiological adherent 3-211 (e.g., biotin) to promote retention of asample. The sample may be retained permanently or temporarily, e.g., forat least a period of time between about 0.5 milliseconds and about 50milliseconds. In another embodiment, the adherent may promote temporaryretention of a sample 3-101for longer periods. Any suitable adherent maybe used in various embodiments, and is not limited to biotin.

According to some embodiments, the layer of material 3-235 adjacent thesample well may be selected based upon an affinity of an adherent forthe material of that layer. In some embodiments, passivation of thesample well's side walls may inhibit coating of an adherent on thesidewalls, so that the adherent 3-211 preferentially deposits at thebase of the sample well. In some embodiments, an adherent coating mayextend up a portion of the sample well's sidewalls. In someimplementations, an adherent may be deposited by an anisotropic physicaldeposition process (e.g., evaporation, sputtering), such that theadherent accumulates at the base of a sample well or divot and does notappreciably form on sidewalls of the sample well.

III. C Sample Well Fabrication

Various fabrication techniques may be employed to fabricate sample wells3-210 for an integrated device. A few example processes are describedbelow, but the invention is not limited to only these examples.

The sample well 3-210 may be formed by any suitable micro- ornano-fabrication process, which may include, but is not limited to,processing steps associated with photolithography, deep-ultravioletphotolithography, immersion photolithography, near-field optical contactphotolithography, EUV lithography, x-ray lithography, nanoimprintlithography, interferometric lithography, step-and-flash lithography,direct-write electron beam lithography, ion beam lithography, ion beammilling, lift-off processing, reactive-ion etching, etc. According tosome embodiments, a sample well 3-210 may be formed usingphotolithography and lift-off processing. Example fabrication stepsassociated with lift-off processing of a sample well are depicted inFIG. 3-9. Although fabrication of only a single sample well or structureat a pixel is typically depicted in the drawings, it will be understoodthat a large number of sample wells or structures may be fabricated on asubstrate (e.g., at each pixel) in parallel.

According to some embodiments, a layer 3-235 (e.g., an oxide layer) on asubstrate may be covered with an anti-reflection (ARC) layer 3-910 andphotoresist 3-920, as depicted in FIG. 3-9A. The photoresist may beexposed and patterned using photolithography and development of theresist. The resist may be developed to remove exposed portions orunexposed portions (depending on the resist type), leaving a pillar3-922 that has a diameter approximately equal to a desired diameter forthe sample well, as depicted in FIG. 3-9B. The height of the pillar maybe greater than a desired depth of the sample well.

The pattern of the pillar 3-922 may be transferred to the ARC layer3-910 via anisotropic, reactive ion etching (RIE), for example as shownin FIG. 3-9C. The region may then be coated with at least one material3-230, e.g., a conductor or metal, that is desired to form the samplewell. A portion of the deposited material, or materials, forms a cap3-232 over the pillar 3-922, as depicted in FIG. 3-9D. The resist andARC may then be stripped from the substrate, using a selective removalprocess (e.g., using a chemical bath with or without agitation whichdissolves at least the resist and releases or “lifts off” the cap). Ifthe ARC remains, it may be stripped from the substrate using a selectiveetch, leaving the sample well 3-210 as shown in FIG. 3-9E. According tosome embodiments, the sidewalls 3-214 of the sample well may be slopeddue to the nature of the deposition of the at least one material 3-230.

As used herein, a “selective etch” means an etching process in which anetchant selectively etches one material that is desired to be removed oretched at a higher rate (e.g., at least twice the rate) than the etchantetches other materials which are not intended to be removed.

Because the resist and ARC are typically polymer based, they areconsidered soft materials which may not be suitable for forming samplewells having high aspect ratios (e.g., aspect ratios greater than about2:1 with respect to height-to-width). For sample wells having higheraspect ratios, a hard material may be included in the lift-off process.For example, before depositing the ARC and photoresist, a layer of ahard (e.g., an inorganic material) may be deposited. In someembodiments, a layer of titanium or silicon nitride may be deposited.The layer of hard material should exhibit preferential etching over thematerial, or materials, 3-230 in which the sample well is formed. Afterthe photoresist is patterned, a pattern of the pillar may be transferredinto the ARC and the underlying hard material 3-930 yielding a structureas depicted in FIG. 3-9F. The photoresist and ARC may be then stripped,the material(s) 3-230 deposited, and a lift-off step performed to formthe sample well.

According to some embodiments, a lift-off process may be used to form asample well comprising energy-enhancing structures 3-711, as depicted inFIG. 3-7C and FIG. 3-7D.

An alternative process for forming a sample well is depicted in FIG.3-10. In this process, the sample well may be directly etched into atleast one material 3-230. For example, at least one material 3-230 inwhich a sample well is to be formed may be deposited on a substrate. Thelayer may be covered by an ARC layer 3-910 and a photoresist 3-920, asillustrated in FIG. 3-10A. The photoresist may be patterned to form ahole having a diameter approximately equal to a desired diameter of thesample well, as depicted in FIG. 3-10B. The pattern of the hole may betransferred to the ARC and through the layer 3-230 using an anisotropic,reactive ion etch, as shown in FIG. 3-10C for example. The resist andARC may be stripped, yielding a sample well as depicted in FIG. 3-10D.According to some embodiments, the sidewalls of a sample well formed byetching into the layer of material 3-230 may be more vertical thansidewalls resulting from a lift-off process.

In some embodiments, the photoresist and ARC may be used to pattern ahard mask (e.g., a silicon nitride or oxide layer, not shown) over thematerial 3-230. The patterned hole may then be transferred to the hardmask, which is then used to transfer the pattern into the layer ofmaterial 3-230. A hard mask may allow greater etching depths into thelayer of material 3-230, so as to form sample wells of higher aspectratio.

It will be appreciated that lift-off processes and direct etchingfabrication techniques described above may be used to form a sample wellwhen multiple layers of different materials are used to form a stack ofmaterial 3-230 in which the sample well is formed. An example stack isshown in FIG. 3-11. According to some embodiments, a stack of materialmay be used to form a sample well to improve coupling of excitationenergy to the excitation region of a sample well, or to reducetransmission or re-radiation of excitation energy into the bulkspecimen. For example, an absorbing layer 3-942 may be deposited over afirst layer 3-940. The first layer may comprise a metal or metal alloy,and the absorbing layer may comprise a material that inhibits surfaceplasmons, e.g., amorphous silicon, TaN, TiN, or Cr. In someimplementations, a surface layer 3-944 may also be deposited topassivate the surface surrounding the sample well (e.g., inhibitadhesion of molecules).

Formation of a divot 3-216 adjacent a sample well may be done in anysuitable manner. In some embodiments, a divot may be formed by etchingfurther into an adjacent layer 3-235, and/or any intervening layer orlayers, adjacent the sample well. For example, after forming a samplewell in a layer of material 3-230, that layer 3-230 may be used as anetch mask for patterning a divot, as depicted in FIG. 3-12. For example,the substrate may be subjected to a selective, anisotropic reactive ionetch so that a divot 3-216 may be etched into adjacent layer 3-235. Forexample, in an embodiment where the material 3-230 is metallic and theadjacent layer 3-235 silicon oxide, a reactive-ion plasma etch having afeed gas comprising CHF₃ or CF₄ may be used to preferentially removeexposed silicon oxide below the sample well and form the divot 3-216. Asused herein, “silicon oxide” generally refers to SiO_(x) and may includesilicon dioxide, for example.

In some embodiments, conditions within the plasma (e.g., bias to thesubstrate and pressure) during an etch may be controlled to determinethe etch profile of the divot. For example, at low pressure (e.g., lessthan about 100 mTorr) and high DC bias (e.g., greater than about 20V),the etching may be highly anisotropic and form substantially straightand vertical sidewalls of the divot, as depicted in the drawing. Athigher pressures and lower bias, the etching may be more isotropicyielding tapered and/or curved sidewalls of the divot. In someimplementations, a wet etch may be used to form the divot, which may besubstantially isotropic and form an approximately spherical divot thatmay extend laterally under the material 3-230, up to or beyond thesidewalls of the sample well.

FIG. 3-13A through FIG. 3-13C depict process steps that may be used toform a divot 3-216 having a smaller transverse dimension than the samplewell 3-210 (for example, a divot like that depicted in FIG. 3-7B). Insome implementations, after forming a sample well, a conformalsacrificial layer 3-960 may be deposited over a region including thesample well. According to some embodiments, the sacrificial layer 3-960may be deposited by a vapor deposition process, e.g., chemical vapordeposition (CVD), plasma-enhanced CVD, or atomic layer deposition (ALD).The sacrificial layer may then be etched back using a first anisotropicetch that is selective to the sacrificial layer 3-960, removes the layerfrom horizontal surfaces, leaves side wall coatings 3-962 on walls ofthe sample well, as depicted in FIG. 3-13B. The etch back may beselective and stop on the material 3-230 and adjacent layer 3-235 insome embodiments, or may be a non-selective, timed etch in someembodiments.

A second anisotropic etch that is selective to the adjacent layer 3-235may be executed to etch a divot 3-216 into the adjacent layer asdepicted in FIG. 3-13C. The sacrificial side wall coatings 3-962 maythen optionally be removed by a selective wet or dry etch. The removalof the sidewall coatings open up the sample well to have a largertransverse dimension than the divot 3-216.

According to some embodiments, the sacrificial layer 3-960 may comprisethe same material as the adjacent layer 3-235. In such embodiments, thesecond etch may remove at least some of the side wall coating 3-962 asthe divot is etched into the adjacent layer 3-235. This etch back of theside wall coating can form tapered sidewalls of the divot in someembodiments.

In some implementations, the sacrificial layer 3-960 may be formed from,or include a layer of, a material that is used to passivate thesidewalls of the sample well (e.g., reduce adhesion of samples at thesidewalls of the sample well). At least some of the layer 3-960 may thenbe left on the walls of the sample well after formation of the divot.

According to some embodiments, the formation of the sidewall coatings3-962 occurs after the formation of the divot. In such an embodiment thelayer 3-960 coats the sidewalls of the divot. Such a process may be usedto passivate the sidewalls of the divot and localize the sample at thecenter of the divot.

Process steps associated with depositing an adherent 3-211 at a base ofa sample well 3-210, and a passivation layer 3-280 are depicted in FIG.3-14. According to some embodiments, a sample well may include a firstpassivation layer 3-280 on walls of the sample well. The firstpassivation layer may be formed, for example, as described above inconnection with FIG. 3-13B or FIG. 3-8. In some embodiments, a firstpassivation layer 3-280 may be formed by any suitable deposition processand etch back. In some embodiments, a first passivation layer may beformed by oxidizing the material 3-230 in which the sample well isformed. For example, the sample well may be formed of aluminum, whichmay be oxidized to create a coating of alumina on sidewalls of thesample well.

An adherent 3-980 or an adherent precursor (e.g., a material whichpreferentially binds an adherent) may be deposited on the substrateusing an anisotropic physical deposition process, e.g., an evaporativedeposition, as depicted in FIG. 3-14A. The adherent or adherentprecursor may form an adherent layer 3-211 at the base of the samplewell, as depicted in FIG. 3-14B, and may coat an upper surface of thematerial 3-230 in which the sample well is formed. A subsequent angled,directional deposition depicted in FIG. 3-14C (sometimes referred to asa shadow deposition or shadow evaporation process) may be used todeposit a second passivation layer 3-280 over an upper surface of thematerial 3-230 without covering the adherent layer 3-211. During theshadow deposition process, the substrate may be rotated around an axisnormal to the substrate, so that the second passivation layer 3-280deposits more uniformly around an upper rim of the sample well. Aresulting structure is depicted in FIG. 3-14D, according to someembodiments. As an alternative to depositing the second passivationlayer, a planarizing etch (e.g., a CMP step) may be used to removeadherent from an upper surface of the material 3-230.

According to some implementations, an adherent layer 3-211 may bedeposited centrally at the base of a tapered sample well, as depicted inFIG. 3-15. For example, an adherent, or adherent precursor, may bedirectionally deposited, as depicted in FIG. 3-14A, in a tapered samplewell, formed as described above. Walls of the sample well may bepassivated by an oxidation process before or after deposition of theadherent layer 3-211. Adherent or precursor remaining on a surface ofthe material 3-230 may be passivated as described in connection withFIG. 3-14D. In some embodiments, an adherent on an upper surface of thematerial 3-230 may be removed by a chemical-mechanical polishing step.By forming an adherent layer, or an adherent layer precursor, centrallyat the base of a sample well, deleterious effects on emission from asample (e.g., suppression or quenching of sample radiation from samplewalls, unfavorable radiation distribution from a sample because it isnot located centrally with respect to energy coupling structures formedaround a sample well, adverse effects on luminescent lifetime for asample) may be reduced.

In some embodiments, lift-off patterning, etching, and depositionprocesses used to form the sample well and divot may be compatible withCMOS processes that are used to form integrated CMOS circuits on anintegrated device. Accordingly, an integrated device may be fabricatedusing conventional CMOS facilities and fabrication techniques, thoughcustom or specialized fabrication facilities may be used in someimplementations.

Variations of the process steps described above may be used to formalternative embodiments of sample wells. For example, a tapered samplewell such as depicted in FIG. 3-7A or FIG. 3-7B may be formed using anangled deposition process depicted in FIG. 3-14C. For the sample well ofFIG. 3-7B, the angle of deposition may be changed during the depositionprocess. For such embodiments, a sample well having substantiallystraight and vertical sidewalls may first be formed, and then additionalmaterial 3-230 deposited by an angled deposition to taper the sidewallsof the sample well.

In some embodiments, a sample well 3-210 may be formed at a pixel afteran excitation source is formed. For example, an excitation source for apixel may be formed at another region and/or at another level on theintegrated device, within or outside a pixel. The type of excitationsource may place processing constraints on the steps used to fabricatethe sample well 3-210. For example, if the excitation source comprisesan organic light-emitting diode (OLED), then processing steps used tofabricate the sample well 3-210 may not exceed temperatures greater thanabout 100° C. Further, the processing steps may not subject the OLED toharsh chemical environments or oxidizing environments.

Any one or more of the foregoing embodiments of sample wells may beincluded in an embodiment of an integrated device.

IV. Excitation Sources

Referring again to FIG. 3-1, there are different types of excitationssources 3-240 that may be used on an integrated device to excite asample 3-101 within a sample well 3-210. According to some embodiments,an excitation source may excite a sample via a radiative process. Forexample, an excitation source may provide visible radiation (e.g.,radiation having a wavelength between about 350 nm and about 750 nm),near-infrared radiation (e.g., radiation having a wavelength betweenabout 0.75 micron and about 1.4 microns), and/or short wavelengthinfrared radiation (e.g., radiation having a wavelength between about1.4 microns and about 3 microns) to at least one excitation region 3-215of at least one sample well. According to some implementations, anexcitation source may provide energy that excites a sample via anon-radiative process. For example, energy may be transferred to asample via Forster resonant energy transfer (FRET) or Dexter energytransfer (DET).

Combinations of energy transfer pathways are also contemplated. Forexample, a radiative excitation source may provide energy to excite anintermediary (e.g., a molecule, a quantum dot, or a layer of materialcomprising selected molecules and/or quantum dots) that is immediatelyadjacent an excitation region of a sample well. The intermediary maytransfer its energy to a sample via a non-radiative process (e.g., viaFRET or DET).

In some embodiments, an excitation source may provide more than onesource of excitation energy. For example, a radiative excitation sourcemay deliver excitation energies having two or more distinct spectralcharacteristics. As an example, a multi-color LED may emit energiescentered at two or more wavelengths, and these energies may be deliveredto an excitation region of a sample well.

IV. A. Radiative Excitation Sources

In overview and according to some embodiments, an integrated device mayinclude at least one radiative excitation source arranged on the deviceto provide excitation energy to at least one excitation region of atleast one sample well or to at least one intermediary that converts orcouples the excitation energy to at least one sample within one or moreexcitation regions. As depicted in FIG. 3-2, radiation 3-247 from anexcitation source 3-240 may impinge on a region around a sample well3-210, for example. In some embodiments, there may beexcitation-coupling structures (not shown) that aid in concentrating theincident excitation energy within an excitation region 3-215 of thesample well.

A radiative excitation source may be characterized by one or moredistinct spectral bands each having a characteristic wavelength. Forinstructional purposes only, an example of spectral emission from anexcitation source is depicted in spectral graph of FIG. 4-1A. Theexcitation energy may be substantially contained within a spectralexcitation band 4-110. A peak wavelength 4-120 of the spectralexcitation band may be used to characterize the excitation energy. Theexcitation energy may also be characterized by a spectral distribution,e.g., a full-width-half-maximum (FWHM) value as shown in the drawing. Anexcitation source producing energy as depicted in FIG. 4-1A, may becharacterized as delivering energy at a wavelength of approximately 540nm radiation and having a FWHM bandwidth of approximately 55 nm.

FIG. 4-1B depicts spectral characteristics of an excitation source (orexcitation sources) that can provide two excitation energy bands to oneor more sample wells. According to some embodiments, a first excitationband 4-112 is at approximately 532 nm, and a second excitation band4-114 is at approximately 638 nm, as illustrated in the drawing. In someembodiments, a first excitation band may be at approximately 638 nm, anda second excitation band may be at approximately 650 nm. In someembodiments, a first excitation band may be at approximately 680 nm, anda second excitation band may be at approximately 690 nm. According tosome embodiments, the peaks of the excitation bands may be within ±5 nmof these values. Other excitation bands may be used in some embodiments.

In some cases, a radiative excitation source may produce a broadexcitation band as depicted in FIG. 4-1A. A broad excitation band 4-110may have a bandwidth greater than approximately 20 nm, according to someembodiments. A broad excitation band may be produced by a light emittingdiode (LED), for example. In some implementations, a radiativeexcitation source may produce a narrow excitation band, as depicted inFIG. 4-1B. A narrow excitation band may be produced by a laser diode,for example, or may be produced by spectrally filtering an output froman LED.

In some embodiments, at least one excitation source 3-240 may be formedat each active pixel 3-100 on a substrate 1-100 of an integrated device,as depicted in FIG. 4-2A. Since a sample well and its excitation regionare small (e.g., having a transverse dimension on the order of 100 nm),an excitation source 3-240 may be formed only in the vicinity of asample well 3-210, as depicted in FIG. 4-2A, according to someembodiments. For example, an excitation source may have a transversedimension that is less than about 20 times a transverse dimension of asample well (e.g., less than about 2 microns in diameter for a samplewell having a diameter of about 100 nm.) Although FIG. 4-2A depicts onlyfour pixels, there may be many more pixels on a substrate 1-100.

In some aspects, each excitation source may be individually controlled.This may require interconnects and drive circuitry (not shown in FIG.4-2A) on the substrate 1-100 for row-and-column addressing of eachsource. The interconnects may run below the pixels and/or in gaps 4-230between pixels 3-100. According to some embodiments, integrated wiringand circuitry may be arranged to control and drive rows or columns ofexcitation sources separately. For example, all excitation sources 3-240in a row may be driven together with a common control signal. In someembodiments, integrated wiring and circuitry may be arranged to controland drive groups of excitation sources on a substrate with a commoncontrol signal. In some implementations, integrated wiring and circuitrymay be arranged to control and drive all excitation sources with acommon control signal. By driving a larger number of excitation sourcestogether, fewer interconnects and drive electronics are required for theexcitation sources.

FIG. 4-2B depicts an embodiment where the excitation sources 4-242 arearranged in strips on a substrate 1-100 of an integrated device. Whenarranged in strips, the excitation sources may exhibit wave guidingproperties, and light from the excitation sources may be guidedlaterally along the strips to the sample wells 3-210. The stripexcitation sources 4-242 may be driven individually in some embodiments,or may be driven together. In some embodiments, the strips may be aarranged in a grid pattern intersecting at the sample wells.

In some implementations, there may be fewer excitation sources formed onan integrated device than pixels, and light from an excitation sourcemay be delivered to more than one sample well via an excitation-couplingstructure such as a waveguide. In some cases, a single excitation sourcemay extend across all active pixels, be controlled by a single drivesignal, and simultaneously illuminate excitation regions in all of thesample wells.

According to some embodiments, excitation sources 4-244 may be arrangedin a checkerboard pattern, as depicted in FIG. 4-2C. In such anembodiment, the excitation sources 4-244 may be located in regions wherethere are no pixels 3-100. Locating excitation sources in regions apartfrom the pixels may ease fabrication constraints for manufacturing anintegrated device. For example, fabrication of the excitation sourcesmay be carried out after fabrication of the sensors and sample wells.Energy from the excitation sources may be provided to the pixels viastrip or slab waveguides (not shown), for example. The excitationsources 4-244 may be driven individually according to some embodiments,in groups according to some embodiments, or all together in someembodiments. Driving excitation sources individually may require agreater number of control circuits and interconnects than may berequired for driving a group of excitation sources.

In some implementations, an excitation source 4-246 may be formed arounda group of pixels 3-100, as depicted in FIG. 4-2D. Radiation from theexcitation source 4-246 may be delivered to the pixels 3-100 via a slabwaveguide or strip waveguides, according to some embodiments. In someimplementations, there may be a reflective wall at the periphery of theexcitation source 4-246 to reflect radiation inward toward the pixels.In some embodiments, there may be integrated circuits 4-210 (e.g., driveelectronic devices, amplifiers, transistors, and/or readout circuitry)located around the periphery of the integrated device on substrate1-100.

By placing the excitation sources in regions where there are no pixels,fabrication of the integrated device may be simplified. For example, afabrication process for the excitation sources 4-244 may besubstantially independent of the fabrication process for the pixels3-100. For example, the excitation sources depicted in FIG. 4-2C andFIG. 4-2D may be fabricated after fabrication of the pixels and/orintegrated circuitry 4-210. This may be desirable when the excitationsource comprises an organic or other material that may be sensitive toor degraded by high process temperatures that may be needed to fabricatethe pixel structures and/or integrated circuits of the integrateddevice.

FIG. 4-2E depicts an embodiment where an excitation source 4-246 ispatterned in a region of the substrate 1-100 adjacent a group of pixels.The drawing depicts an elevation view of both the excitation source4-246 and a sample well 3-210 that may be located more than 100 micronsfrom the excitation source. The sample well may be located within anypixel within a group of pixels on the integrated device. The excitationsource 4-246 may comprise an edge-emitting light emitting diode (LED) insome implementations, or laser diode in some embodiments, and comprise adiode stack 4-250. Electrical connection to the diode stack may includea cathode pad 4-281 and an anode pad 4-282. The cathode and anode may bemetallic and reflective and may include scattering structures to reflectand/or scatter radiation laterally.

According to some embodiments, light from the diode 4-246 may coupleinto a slab waveguide that guides the radiation to the sample wellswithin a group of pixels. The slab waveguide may comprise a firstdielectric layer 3-235, a core layer 4-270, and a second dielectriclayer 3-245. The refractive index of the core layer 4-270 may be greaterthan the refractive index of the first and second dielectric layers. Forexample, the core layer may comprise a silicon nitride layer, and thefirst and second dielectric layers may comprise silicon oxide layers.Light from the excitation source 4-246 may be substantially confined tothe core dielectric layer 4-270, and guided to the sample wells 3-210.In some embodiments, a divot of the sample well may extend to, partwaythrough, or fully through the core layer 4-270. In some embodiments, thecore layer may serve as an etch stop during etching of the divots at thesample wells.

Although the embodiment depicted in FIG. 4-2E is described in connectionwith the distributed excitation source 4-246 as depicted in FIG. 4-2D,in other embodiments discrete excitation sources may be located at theperiphery of a group of pixels and delivered to at least some pixels(e.g., pixels in one or more rows or columns) via strip waveguidesrather than a slab waveguide.

Various types of radiative excitation sources are depicted in FIG. 4-3Athrough FIG. 4-3E. According to some embodiments, an excitation sourcemay comprise an organic light emitting diode (OLED). An OLED maycomprise an organic emissive layer 4-342 and an organic conducting layer4-344, as depicted in FIG. 4-3A. Each organic layer may comprise organicmolecules and/or be formed of an organic polymer. Organic molecules inthe emissive layer may be selected to emit at a desired wavelength or acombination of desired wavelengths. Electrical contact to the OLED maybe made through a cathode and an anode 4-346. The anode may be formed ofany suitable conductive material, and may be formed prior to depositionof the organic layers. The anode 4-346 may include an opening 4-347adjacent the sample well, so that emission from a sample may passthrough the substrate to a sensor located below the sample, according tosome embodiments. In some embodiments, the cathode may compriseconductive material 3-230 or a conductive layer in which the sample well3-210 is formed. In some implementations, a divot 3-216 may be formedinto the emissive layer 4-342 of the OLED, so that light from the OLEDmay be delivered more efficiently into the excitation region of thesample well.

In some implementations, an OLED may be vertically spaced farther from asample well than is shown in FIG. 4-3A. For example, an OLED may beformed below an insulating or transparent layer 3-235. In suchembodiments, a cathode of the OLED may comprise a transparent conductor,such as indium tin oxide (ITO), so that light from the OLED may passthrough to the sample well. According to some embodiments, an OLED maybe spaced below a sample well between 500 nm and 10 microns.

One advantage of using an OLED for the integrated device is that modernOLEDs are capable of high light intensity output. Another advantage is alow cost of OLEDs. Issues associated with OLED lifetime are notproblematic for an integrated device, since an integrated device may beused one or a few times, and may be disposed before there is sufficientuse of the OLED to degrade its performance

Solid-state or semiconductor LEDs may also be used to illuminate samplewells according to some embodiments. FIG. 4-3B depicts an integratedsemiconductor light emitting diode that may be fabricated adjacent to asample well, in some implementations. A semiconductor LED may comprise aplurality of layers, as depicted in the drawing. The layers may includean electron transport layer 4-352, a hole blocking layer 4-354, andemissive layer 4-356, a hole transport layer 4-357, and an electronblocking layer 4-359, in some implementations. The stack of layers maybe electrically contacted by an anode and cathode as described inconnection with FIG. 4-3A. The LED structure depicted in FIG. 4-3B maybe used for other types of LEDs, including but not limited to OLEDs,PhOLEDs, and a quantum dot LEDs (QLEDs).

According to some embodiments, a semiconductor laser diode may beintegrated onto the substrate 1-100. FIG. 4-3C depicts a vertical cavitysurface emitting laser (VCSEL) that may be used in some implementations.A VCSEL may comprise reflective stacks 4-364, 4-362 formed at oppositeends of a VCSEL cavity. A multiple quantum well 4-365 may be formedwithin the VCSEL's cavity. In some implementations, a reflectivematerial 3-230 may form a cathode or anode at one end of the VCSELcavity, and a sample well 3-210 may be formed in the cathode or anode.According to some embodiments, a divot 3-216 may extend into the cavityof the VCSEL, as shown in the drawing.

Since the divot extends into the VCSEL resonator, the sample may beexposed to appreciably higher intensity than it would if it were locatedoutside the cavity. For example, if the reflective stacks of the VCSELcavity are greater than 90%, then the intensity within the cavity may bebetween approximately 10 and 100 times higher than the intensity outsidethe cavity. In some embodiments, at least one reflector of the VCSELcavity may be dichroic, such that it is highly reflective for theexcitation energy (e.g., greater than about 90%) and transmits a highpercentage of emission from the sample (e.g., more than about 60%).

In FIG. 4-3B and FIG. 4-3C, the excitation sources extend across an areaappreciably larger than the transverse dimension of the sample well.Accordingly, emission from a sample must pass through the excitationsource to reach a sensor located below the sample well. Some of theemission from a sample may be absorbed within the excitation source insuch embodiments.

FIG. 4-3D and FIG. 4-3E depict nanoscale excitation sources havingtransverse dimensions approximately equal to the transverse dimension ofthe sample well. For example, a transverse dimension of a nanoscaleexcitation source may be between 50 nm and 500 nm, according to someembodiments, though may be larger in other embodiments. These nanoscaleexcitation sources may be self-aligned to the sample well duringfabrication, according to some embodiments. In some embodiments,microscale excitation sources may be formed adjacent the sample well,similar to the nanoscale excitation sources, but having microscaletransverse dimensions.

FIG. 4-3D depicts a nano-LED that is formed below a sample well. Thenano-LED may comprise a pillar 4-374 having a first type of conductivityand a cap 4-376 having a second type of conductivity to form a p-njunction. The pillar 4-374 may be formed by epitaxial growth from asemiconductor layer 4-380. During growth of the pillar, a reflectivestack 4-375 may be formed by alternating materials of the pillar and/ordopant concentration, according to some embodiments. Electrical contactto the pillar 4-374 may be made through the semiconductor layer 4-380.The nano-LED may further comprise a conductive surface coating 4-372that is used to electrically connect to the cap 4-376 via a conductivematerial 3-230 in which the sample well is formed. In some embodiments,there may be more layers in the nano-LED than shown in the drawing. Forexample, the nano-LED may include electron transport, electron blocking,hole transport, and/or hole blocking layers. A passivating layer 4-378(e.g., an oxide) may be deposited over a region around and within thesample well, according to some embodiments.

In some embodiments, the passivating layer 4-378 in the conductive layer4-372 may be transparent to radiation emitted by a sample in the samplewell. For example, the passivating layer 4-378 may comprise alumina oran oxide. The conductive coating 4-372 may also be transparent toradiation emitted by a sample, and may comprise indium tin oxide (ITO),according to some embodiments. In some implementations, the conductivecoating 4-372 may comprise graphene, indium-doped zinc oxide,aluminum-doped zinc oxide, or gallium-doped zinc oxide. Because thepassivating layer and conductive coating are transparent, light from asample may travel to the semiconductor substrate 4-380 without passingthrough silicon or semiconductor, as may occur in the devices depictedin FIG. 4-3B and FIG. 4-3C. In some embodiments, one or more sensors(not shown) may be formed in the substrate 4-380 to detect emission fromthe sample.

According to some embodiments, a nano-LED 4-371 or nanoscale excitationsource may comprise a vertical waveguide. For example, the refractiveindex of the nano-LED may be appreciably greater than the refractiveindex of the surrounding layer 3-235. Accordingly, emission from thenano-LED 4-371 may be vertically guided to and concentrated at anexcitation region of a sample well 3-210. As such, the nano-LED may moreefficiently illuminate the excitation region than a larger device, suchas the diode depicted in FIG. 4-3B, for example.

In some embodiments, the cap 4-376 having a second type of conductivityof the nano-LED may be etched back to expose the pillar. A reflectivecoating may then be formed on an upper surface, opposite the reflectivestack 4-375 to form a nanoscale, vertical edge emitting laser diode(nano-VEELD).

The height of the nano-LED may be carefully controlled by epitaxialgrowth. Accordingly, a distance between an emitting end of the nano-LEDand a lower surface of the material 3-230, in which the sample well isformed, may be carefully controlled. Additionally, a directionalphysical deposition (such as described in connection with FIG. 3-9D) ofa passivating material or dielectric may be used to carefully control adistance between an excitation region of the sample well and theemitting end of the nano-LED. Careful control of these distances incombination with coupling structures formed adjacent the sample well mayimprove coupling of excitation energy into the excitation region andcoupling of emission from the sample to one or more sensors.

FIG. 4-3E depicts a self-aligned, nano-VCSEL formed below a sample well,according to some implementations. The nano-VCSEL may be formed usingsimilar techniques to those used to form the nano-LED (described infurther detail below). According to some embodiments, the nano-VCSEL maycomprise a first reflective stack 4-377, a multiple quantum wellstructure 4-330 or quantum dot, and a second reflective stack 4-373formed during epitaxial growth of the pillar. Electrical connection toone end of the nano-VCSEL may be made by a conductive coating 4-372, asdescribed in connection with the nano-LED.

Because of their small sizes, the nano-LED 4-371 and nano-VCSEL willhave low junction capacitances. Accordingly, they may be modulated athigh speeds. In some embodiments, a nano-LED, nano-VEELD, or nano-VCSELmay have turn-on and turn-off times less than approximately 1microsecond. A nano-LED, nano-VEELD, or nano-VCSEL may have turn-on andturn-off times less than approximately 100 nanoseconds in someimplementations, less than approximately 10 nanoseconds in someimplementations, less than approximately 1 nanosecond in someembodiments, and yet less than approximately 100 picoseconds in someembodiments.

Additionally, since intense excitation energy is only needed at theexcitation region of the sample well, a nano-LED, nano-VEELD, ornano-VCSEL may more efficiently excite a sample with less total outputpower. This may be beneficial in some implementations, since high powerdissipation may heat a specimen to unacceptably high temperatures andpossibly damage the sample. Further, since the emission from thenano-LED, nano-VEELD, or nano-VCSEL is delivered primarily only to theexcitation region, less overall excitation energy is required, ascompared to a larger excitation source emitting radiation over an areasubstantially larger than the excitation region 3-215 of a sample well3-210. Because less overall excitation energy is required, thesignal-to-noise ratio for emission from the sample may increase due toless background radiation from the excitation source, according to someembodiments.

Various techniques may be used to fabricate the excitation sourcesdescribed in FIG. 4-3A through FIG. 4-3D. According to some embodiments,conventional techniques may be used to form at least one OLED, PhOLED,or QLED on an integrated device. For example, multiple layer depositionsmay be carried out at regions where the LEDs are formed, while otherregions may be masked off to prevent depositions on the substrate atthose regions. Because an OLED, PhOLED, or QLED device may be sensitiveto elevated temperatures, processing temperatures after formation ofsuch devices may need to be kept below a temperature limit, above whichdamage may occur to the OLED, PhOLED, or QLED devices. For example, anOLED may be damaged or degraded by exposure to temperatures aboveapproximately 100° C. Accordingly, after formation of an OLED on anintegrated device, processing temperatures may be limited toapproximately 100° C. during subsequent fabrication steps.

Formation of inorganic semiconductor LEDs, laser diodes, or VCSEL's onan integrated device may be carried out using conventional techniques(e.g., using ion implantations and diffusions and/or multiple epitaxialdepositions), according to some embodiments. In other implementations,inorganic semiconductor LEDs, laser diodes, VCSELs, and/or sample wellsmay be formed on a separate semiconductor-on-insulator (SOI) substratethat is subsequently aligned and bonded to the substrate 1-100 of theintegrated device. For example, epitaxial layers for a laser diode orVCSEL may be formed on an SOI substrate, which can then be bonded to adielectric layer 3-235 depicted in FIG. 4-3B, for example. Afterbonding, the silicon layer having the formed VCSEL may be released fromthe SOI substrate, and may further be etched back according to someembodiments. Sample wells 3-210 may then be formed at each pixel.

IV. B. Fabrication of Radiative Excitation Sources

FIG. 4-4A through FIG. 4-4I depict structures associated with processsteps that may be used to form a nano-LED, nano-VEELD, or nano-VCSELthat is self-aligned to a sample well, according to some embodiments.The depicted process steps illustrate only some embodiments of methodsthat may be used to fabricate the devices. The nano-LED, nano-VEELD, ornano-VCSEL devices may be fabricated using other or additional processsteps in some embodiments. For example, some processes may requirephotolithographic alignment of these devices to the sample wells. Insome embodiments, microscale excitation sources may be fabricated usingone or more steps described for fabrication of nanoscale excitationsources.

According to some implementations, a hole or via 4-410 may be formed ina substrate comprising a semiconductor layer 4-380 an insulating layer3-235 and a top layer 3-230, as depicted in FIG. 4-4A. The hole may beformed by patterning a hole in resist over the top layer 3-230, asdescribed in connection with FIG. 3-10A through FIG. 3-10D. A firstselective anisotropic etch may be used to etch the hole pattern throughthe top layer, and a second selective anisotropic etch may be used toetch the hole pattern into the insulating layer 3-235.

The top layer may comprise a material or stack of materials in which thesample well is formed, and the hole 4-410 may define the location of asample well. In some embodiments, the semiconductor layer 4-380 maycomprise silicon, though other semiconductor materials may be used. Insome cases, the semiconductor layer 4-380 may comprise a thin, orultrathin (e.g., less than approximately 50 nm thick), semiconductorlayer of a SOI substrate, such that emission from the sample well maypass through the layer with less than about 30% loss. The insulatinglayer 3-235 may comprise an oxide (e.g., SiO₂) or any suitable materialthat transmits radiation from the excitation source and emission fromthe sample. The top layer 3-230 may comprise a conductive metal,according to some implementations, a stack of materials (e.g., asemiconductor, a metal, and an insulator), or any suitable combinationof materials described herein in connection with fabrication of thesample well 3-210.

A sacrificial coating 4-420 may then be deposited to line the hole4-410, as depicted in FIG. 4-4B. The sacrificial coating 4-420 may firstbe deposited over the region is a uniform layer (not shown). Forexample, the sacrificial coating may be deposited using a conformaldeposition process, such as a chemical vapor deposition (CVD) process oran atomic layer deposition (ALD) process. After deposition, thesacrificial coating may be etched back to remove the sacrificialmaterial on the horizontal planar surfaces (as viewed in the drawing).Such a deposition and etch-back process is described in connection withFIG. 3-13A and FIG. 3-13 B. Any suitable material may be used for thesacrificial coating 4-420. In various embodiments, the material used forthe sacrificial coating 4-420 will exhibit etch selectivity over the toplayer 3-230, the insulating layer 3-235, and a semiconductor materialthat will be epitaxially grown from the semiconductor layer 4-380. Insome embodiments, the sacrificial coating 4-420 may be formed fromsilicon nitride, for example. After formation of the sacrificial coating4-420, a second hole 4-412 may be etched to the semiconductor layer4-380 to expose a surface of the semiconductor layer. The resultingstructure may appear as depicted in FIG. 4-4C, according to someimplementations.

A semiconductor pillar 4-374 may then be grown from the semiconductorlayer 4-380, as depicted in FIG. 4-4D. According to some embodiments,the pillar may be grown via an epitaxial growth process, such asmolecular organic chemical vapor deposition (MOCVD). According to someembodiments, the pillar may form in the hole that was etched to thesemiconductor layer and is defined by walls of the insulating layer3-235 and the sacrificial coating 4-420. The hole may provide a mold forthe growth of the pillar. Since a sample well 3-210 will be subsequentlyformed in an upper portion of the hoe, the pillar 4-374 growsself-aligned to the subsequently formed sample well 3-210.

In some embodiments, during growth of the pillar, a reflective stack4-375 may, or may not, be formed at the base of the pillar. Thereflective stack may exhibit a high reflectivity for emission from thenano-LED, according to some implementations, and may be used to reflectapproximately one-half of the emission from the nano-LED toward thesample well. In some embodiments, the reflective stack 4-375 may exhibita low reflectivity (e.g., less than about 30%) for emission from asample in the sample well.

The pillar 4-374 may be formed with any selected conductivity type. Forexample, donor or acceptors species may be added during epitaxial growthof the pillar to define the conductivity type of the pillar. In someembodiments the pillar may be p type, and in other embodiments thepillar may be n type.

After formation of the pillar 4-374, the sacrificial coating 4-420 maybe removed by a selective etching process. The etching process may be awet etch or a dry etch that preferentially removes the sacrificialcoating but does not appreciably remove the insulating layer 3-235 toplayer 3-230 or semiconductor pillar 4-374. Removal of the sacrificialcoating 4-420 leaves an upper portion of the semiconductor pillarexposed within the hole.

A second epitaxial growth may then be executed to form a semiconductorcap 4-376 over the pillar, as depicted in FIG. 4-4E. The conductivitytype of the semiconductor cap 4-376 may be made opposite theconductivity type of the pillar 4-374 to form a p-n junction. In someembodiments, the semiconductor cap 4-376 may fill a mid-region of thehole 4-410 as it grows from the exposed pillar. In some implementations,the semiconductor cap may not entirely fill the lower region of thehole, and may leave an open space between it and sidewalls of insulatinglayer 3-235.

A conductive surface layer 4-372 may then be deposited over the region,as depicted in FIG. 4-4E. The conductive surface layer may comprise alayer of ITO in some embodiments, and provide electrical connection fromthe top layer 3-230 to the semiconductor cap 4-376 (e.g., an electricalconnection to a p or n region of the nano-LED). The conductive surfacelayer 4-372 may be deposited by any suitable conformal depositionprocess, e.g., atomic layer deposition or chemical vapor deposition,according to some embodiments.

In some implementations, a passivating layer 4-378 may then be depositedconformably over the region, as depicted in FIG. 4-4F. The passivationlayer may be an insulating layer according to some embodiments, such asalumina or silicon oxide. An adherent (not shown) may be deposited at abase of the sample well, as described in connection with FIG. 3-14 orFIG. 3-15, for example. As can be seen in FIG. 4-4F, the resultingsample well 3-210 and nano-LED are self-aligned.

Structures associated with process steps for alternative embodiments offabricating a nano-LED are shown in FIG. 4-4G through FIG. 4-4I. Afterobtaining a structure depicted in FIG. 4-4B, for example, selectiveanisotropic etching steps may be executed to selectively removehorizontal planar surfaces of the coating layer 4-372 and a portion ofthe semiconductor cap 4-376 and semiconductor pillar 4-374. Theresulting structure may appear as depicted in FIG. 4-4G.

In some embodiments, a spacing layer 4-440 may then be deposited overthe region. The spacing layer may comprise a transparent material, forexample, silicon oxide. The spacing layer may be deposited by a physicaldeposition process, e.g., electron beam evaporation. The spacing layermay form a plug 4-442 at the bottom of the sample well. The plug may beused to carefully space the location of a sample from an end of thenano-LED. Since the thickness of a deposited layer can be controlledvery accurately, to within a few nanometers, the spacing between an endof the nano-LED and the location of the sample can be controlled quiteprecisely. A passivation layer 4-378 may then be deposited over theregion, as depicted in FIG. 4-4I, and an adherent subsequentlydeposited.

Some process steps used to form a self-aligned, nano-LED may also beused to fabricate a self-aligned nano-VEELD or self-aligned nano-VCSEL.Fabrication of a nano-VCSEL may not require steps depicted in FIG. 4-4Band FIG. 4-4C. Instead, the hole 4-410 may be etched to thesemiconductor layer 4-380, and a first portion of the VCSEL pillar(e.g., a p-type portion), including a reflective stack 4-375, may beformed in the hole by epitaxial growth. Subsequently, an n-type portionof the pillar may be formed, and electrical contact made to the n-typeportion using a step as depicted in FIG. 4-4E, for example.

According to some implementations, a thickness of the insulating layer3-235 for forming a nano-LED, nano-VEELD, or nano-VCSEL may be betweenapproximately 100 nm and approximately 2 microns. In someimplementations, one or more sensors 3-260 (not shown in the drawings)may be patterned in the semiconductor layer 4-380 to detect emissionfrom the sample well 3-210. The one or more sensors may be patternednear the nanoscale excitation source, so that the excitation source,sample well, and sensor are contained within a volume measuring lessthan about 20 microns in a maximum transverse dimension and less thanabout 2 microns in height. In some embodiments, the volume may be lessthan about 5 microns in a maximum transverse dimension.

If greater photon flux is needed, a nano-LED, nano-VEELD, or nano-VCSELmay be fabricated to have a transverse dimension larger than atransverse dimension of the sample well, using process steps similar tothose depicted in FIG. 3-13A and FIG. 3-13B after formation of thenanoscale excitation source.

IV. C. Non-Radiative Excitation Sources

Samples 3-101 in an excitation region of a sample well may be excitedvia non-radiative processes, according to some embodiments. Anon-radiative process may include Forster resonant energy transfer(FRET), which may occur over distances up to about 10 nm, or Dexterenergy transfer (DET), which may occur over distances up to about 1 nm.Accordingly, non-radiative excitation sources that may be included in anintegrated device have also been contemplated by the inventors. As withthe radiative excitation sources, there may be one or moreseparately-controllable, non-radiative excitation sources on anintegrated device. For example, in some embodiments a singlenon-radiative source may be shared by a group of pixels or an entirepixel array of an integrated device. In some implementations, anon-radiative excitation source may be fabricated at each pixel.

FIG. 4-5A depicts just one embodiment of a non-radiative excitationsource that may be formed at a pixel of an integrated device. Accordingto some embodiments, a semiconductor layer 4-510 may be formed on aninsulating substrate 4-530. The semiconductor layer may comprise anorganic semiconductor or an inorganic semiconductor. In someimplementations, the semiconductor layer may be a thin, or ultrathin,semiconductor layer of an SOI substrate. The semiconductor layer mayhave a thickness between approximately 10 nm and approximately 100 nm,according to some embodiments. First electrodes 4-520 may be disposed onthe semiconductor layer. The electrodes 4-520 may run along a surface ofthe semiconductor layer 4-510, e.g., extending along spaces betweensample wells 3-210, and provide a first electrical connection to thesemiconductor layer. A second insulating layer 4-540 may be formedadjacent the semiconductor layer and the first electrodes 4-520. Aconductive layer 4-542 may be formed adjacent the second insulatinglayer 4-540. A sample well 3-210 may be formed in the second insulatinglayer and in the conductive layer 4-542, according to some embodiments,and a conductive coating 4-544 may be formed on walls of the samplewell, as depicted in the drawing. The conductive coating 4-544 mayprovide a second electrical contact to the semiconductor layer.

In operation, and electrical bias may be applied between the firstelectrodes 4-520 and the conductive layer 4-542. A current may flowthrough the semiconducting layer 4-510 near the sample well, andgenerate excitons 4-508 within the semiconductor layer 5-510. Theexcitons may be generated by collisional excitation, according to someembodiments, and may diffuse to the surface of the semiconductor layer4-510 at the sample well 3-210. When near the surface at the samplewell, the excitons may deliver energy to a sample within the sample wellvia FRET or DET.

An alternative embodiment of a non-radiative excitation source isdepicted in FIG. 4-5B. According to some embodiments, a non-radiativeexcitation source may comprise a lateral diode 4-512 formed adjacent abase of sample well 3-210. In some embodiments, the diode may comprisep-n junction or may comprise an intrinsic or undoped region 4-513 toform a p-i-n junction. A non-radiative excitation source as depicted inFIG. 4-5B may include cathode and anode electrodes 4-520 for makingelectrical contact to the p and n regions of the diode. In someembodiments, there may be a thin passivating layer (e.g., less than 10nm) and/or adherent layer 4-550 formed at the junction region of thediode.

A plan view of one embodiment of a non-radiative excitation sourcedepicted in FIG. 4-5B is shown in FIG. 4-5C, according to someembodiments. The plan view is taken at the an interface between thefirst insulating layer 4-530 and the second insulating layer 4-540. Theplan view depicts just one embodiment of how the p and n regions of thediode and electrodes may be arranged on the integrated device. In someembodiments, heavily doped regions of semiconductor 4-515, 4-517 may beformed near the electrodes, and may extend to a region near each samplewell. Any suitable pattern may be used to arrange the p and n regions ofthe diodes in an integrated device. For example, instead of using aninterdigitated pattern as depicted in FIG. 4-5C, a serpentine pattern ofthe diodes may be used instead.

FIG. 4-5D depicts in an alternative embodiment of a non-radiativeexcitation source that may be formed by vertical growth at a sample well3-210. In some implementations, the source comprises a diode 4-514 thatis self-aligned to the sample well 3-210. The diode may comprise ananoscale p-n or p-i-n diode having a cylindrical pillar formed of asemiconductor having a first type of conductivity that is surrounded bya semiconductor cylindrical shell having a second type of conductivity,as depicted in the drawing. The shape of the nano-diode may be anysuitable shape and need not be cylindrical. For example, if the samplewell is formed to have a square, rectangular, or polygonalcross-section, then the nano-diode may assume a similar shape.

A connection to a first region (e.g., an n region) of the diode may bemade through a semiconductor layer 4-380. In some embodiments, aheavily-doped well 4-582 may be formed on the semiconductor layer at abase of the nano-diode to improve electrical connection to the diode. Aconnection to the second region of the diode may be made through aconductive layer 3-230, in which the sample well is formed, and aconductive coating 4-372. In some embodiments, the conductive coating4-372 may comprise ITO.

In operation, electrical current within the nano-diode may generateexcitons that diffuse to the surface of the diode where a majorityrecombines. In some cases, excitons may transfer energy non-radiativelyto a sample within the sample well 3-210 upon recombination.Accordingly, sample excitation within a sample well may occur withoutradiative emission from an excitation source. A benefit of non-radiativeexcitation is that the excitation source may contribute no appreciableradiation noise at the sensor during signal detection. A second benefitis that sample excitation is localized to within about 10 nm from theexcitation source. This may be beneficial for coupling energy from thesample to the sensor, and may also reduce or eliminate noise radiationfrom other samples in the specimen that are more than about 10 nm fromthe excitation source.

Various fabrication techniques may be used to fabricate non-radiativesources depicted in FIG. 4-5A through FIG. 4-5D. Fabrication of a devicedepicted in FIG. 4-5A may employ conventional patterning and etchingprocess steps, and may include process steps associated with forming asample well as described herein. In some embodiments, a nano-diode asdepicted in FIG. 4-5A may be formed employing fabrication steps that areused to form the nano-LED, as described in connection with FIG. 4-4G.

IV. D. Fabrication of Non-Radiative Excitation Sources

FIG. 4-6A through FIG. 4-6U depict structures associated with processsteps that may be used to form a lateral-junction, non-radiativeexcitation source that is self-aligned to a sample well, as depicted inFIG. 4-5B, for example. The intrinsic region, or p-n junction may beself-aligned to a base of the sample well.

According to some embodiments, the process may begin with obtaining asilicon on insulator substrate as depicted in FIG. 4-6A, comprising asemiconductor substrate 4-535, an insulating layer 4-530, and anintrinsic or undoped semiconductor layer 4-513. In some embodiments,integrated circuitry may be formed in the semiconductor substrate 4-535,such as one or more sensors used to detect emission from a sample andassociated integrated circuits that may be used in the integrateddevice. The insulating layer 4-530 may have a thickness between about 50nm and about 500 nm, according to some embodiments, though otherthicknesses may be used in other embodiments. The semiconducting layer4-513 may be disposed on the insulating layer and have a thicknessbetween approximately 10 nm and approximately 100 nm.

A mask 4-610 comprising bars may be patterned on the semiconductinglayer 4-513, as depicted in FIG. 4-6B. The bars may run along thesurface of the semiconducting layer, and may have the appearance of agrating. The mask may be a hard mask in some embodiments, for example amask formed from silicon oxide, though other materials may be used. Inother embodiments a soft mask, e.g., formed from a polymer, may be used.The mask may exhibit etch selectivity over the semiconductor layer4-513. A thickness of the mask may be between approximately 50 nm andapproximately 250 nm. A spacing between the bars of the mask may be onthe order of the separation distance between pixels of the integrateddevice. A width of the bars may also be on the order of the separationdistance between pixels of the integrated device. The bars may extendacross the active pixel region of the integrated device. In someimplementations, the mask 4-610 may be aligned to the substrate suchthat edges of the bars are approximately centered over sensors formed onthe semiconductor substrate 4-535.

In some embodiments, a layer 4-620 may be conformably deposited over themask 4-610, as depicted in FIG. 4-6C. The layer may comprise a hardmaterial, such as silicon nitride, according to some embodiments. Thelayer 4-620 may exhibit etch selectivity over the semiconductor layer4-513 and over the mask 4-610, according to some embodiments. Athickness of the layer 4-620 may be approximately equal to a desiredsize of the sample well 3-210, in some implementations. For example, athickness of the layer 4-620 may be between approximately 80 nm andapproximately 250 nm, though other thicknesses may be used in someembodiments.

The layer 4-620 may then be etched back using a selective anisotropicetch process, yielding the structure as shown in FIG. 4-6D. The etch ofthe layer 4-620 removes horizontal portions of the layer and leaves thevertical sidewalls 4-622 adjacent the mask bars 4-610. The region of thesubstrate may then be subjected to ion implantation, as depicted in thedrawing. For example donor or acceptor ions may be implanted into thesemiconducting layer 4-513 where the layer is exposed. The ions may beblocked from entering the semiconductor layer by the vertical sidewalls4-622 and the mask 4-610. In some embodiments, the ion implantation maycomprise donors and produce n-type semiconductor regions 4-632, asdepicted in FIG. 4-6E. The regions under the vertical sidewalls and themask may remain intrinsic regions of semiconductor 4-630.

A thin layer 4-624 may then be conformably deposited over the region, asdepicted in FIG. 4-6F. According to some embodiments, the thin layer maybe formed of a same material as the layer 4-620. According to someembodiments the thin layer may be silicon nitride, though othermaterials may be used in other embodiments. A thickness of the layer4-624 may be between approximately 5 nm and approximately 20 nm,according to some embodiments.

A planarizing material 4-640 may then be deposited over the region, asdepicted in FIG. 4-6G. According to some embodiments, the planarizingmaterial 4-640 exhibits etch selectivity over the mask 4-610, thevertical sidewalls 4-622, and the thin layer 4-624. In some embodiments,the planarizing material may further exhibit etch selectivity over theintrinsic region of the semiconductor layer 4-630. According to someimplementations, the planarizing material 4-640 may comprise amorphoussilicon, though other materials may be used in other embodiments.

The material 4-640 and surface of the substrate may then be planarized,as depicted in FIG. 4-6H. For example, a chemical mechanical polishing(CMP) step may be used to planarize the region. The CMP step mayselectively etch the material 4-640 but not etch the layer 4-624, andessentially stop on the layer 4-624, in some implementations. Anonselective planarizing etch may then be used to etch back thesubstrate to expose the mask bars 4-610, as depicted in FIG. 4-6I.

The mask bars 4-610 may then be removed by a selective etching process.The selective etching process may be a dry etch or a wet etch. Theresulting structure may appear as depicted in FIG. 4-6J. The substratemay then be subjected to a second ion implantation as indicated in thedrawing. For example, acceptor ions may be implanted into the exposedregions of the intrinsic semiconductor layer 4-512, converting theseregions to p-type semiconductor regions 4-634. The planarizing material4-640 may then be removed by a selective dry or wet etch.

In some embodiments, an additional planarizing polymer or oxide layer(not shown) may be formed on the substrate to protect the p-type regions4-634 and planarized to expose the planarizing material 4-640, beforeremoving the planarizing material 4-640. After removal of theplanarizing material 4-640, the additional planarizing material may beselectively etched leaving portions of the vertical sidewalls 4-622 andthe remaining portion of the thin layer 4-624.

When the vertical sidewalls 4-622 and the thin layer 4-624 are exposed,they may be etched back with an anisotropic selective etch to remove thehorizontal portion of the thin layer 4-624 covering the n-type regions4-632. The resulting structure may appear as depicted in FIG. 4-6K, andshows remaining vertical bars 4-626 that comprise remaining portions ofthe vertical sidewalls 4-622 and remaining portion of the thin layer4-624. Undoped, intrinsic regions 4-630 of the semiconductor layerremain under the vertical bars 4-626. In some embodiments, a thermaldiffusion process, e.g., a spike anneal, may be used to drive dopantsunder the vertical bars to reduce the spatial extent of a p-i-njunction, or if a p-n junction is preferred instead of a p-i-n junction.

A resist 4-650 (e.g., a photoresist) may be deposited over the region,according to some embodiments, as indicated in FIG. 4-6L. The resist maybe patterned using a mask that is aligned to the vertical bars 4-626,such that portions of the resist over the n- and p-type regions of thelateral diode structure are exposed and developed away. Electrodes 4-520may then be formed using a lift-off process, according to someembodiments. For example, electrode material may be deposited on theexposed n- and p-type regions of the diode. The remaining resist may bestripped from the substrate lifting off portions of the electrodematerial on top of the resist 4-650, leaving the vertical bars andelectrodes over the lateral p-i-n regions.

A planarizing material 4-660 may then be deposited over the region andthe material and region planarized, as depicted in FIG. 4-6M. Theplanarizing material 4-660 may be an oxide in some embodiments orpolymer in some embodiments. In some implementations, a CMP step may beused to planarize the region and expose the vertical bars 4-626. FIG.4-6N depicts a plan view of the region after planization, according tosome embodiments. The dashed line indicates the location of across-section corresponding to the elevation view shown in FIG. 4-6M.

Additional masking bars 4-670 may then be patterned on top of the activepixel region, as depicted in the plan view of FIG. 4-60. The bars may bepatterned using any suitable lithography process, and may have a widththat is approximately equal to a width of the vertical bars 4-626. Themasking bars 4-670 may be oriented transverse to the vertical bars4-626, and the masking bars may be spaced apart a distance that isapproximately equal to a distance between pixels of the integrateddevice. In various embodiments, the masking bars are aligned to centersof sensors that may be located in the semiconductor substrate 4-535below the vertical bars 4-626. According to some embodiments the maskingbars 4-670 exhibit etch selectivity over the vertical bars 4-626. Forexample, the mask bars may be formed of a polymer or an oxide, and avertical bars may comprise silicon nitride.

As indicated in FIG. 4-6P, a selective anisotropic etch may be used toetch away portions of the vertical bars 4-626 to expose the underlyingintrinsic regions 4-630 of the semiconductor layer. Because of themasking bars 4-670, portions of the vertical bars 4-626 remainunderneath the mask bars.

The masking bars 4-670 may then be removed from the region, and aplanarizing layer 4-680 (e.g., an oxide layer) deposited over theregion. A CMP step may be used to planarize the pixel region yielding astructure as depicted in FIG. 4-6Q, in plan view, and FIG. 4-6R, inelevation view. The remaining portions of the vertical bars formvertical pillars 4-628, according to some embodiments. In someimplementations, a transverse dimension of the vertical pillars 4-628 isbetween approximately 80 nm and approximately 250 nm. The verticalpillars may have an approximately square or rectangular cross-sectionalshape. The pillars 4-628 may then be selectively etched and remove fromthe substrate to produce sample wells that are substantiallyself-aligned to the underlying diode junction, as depicted in FIG. 4-6S.

As depicted in FIG. 4-6Q, a spacing between the pillars 4-628 may beapproximately equivalent in a first direction (a vertical direction asviewed on the page). The spacing between the pillars may not beequivalent in the second direction (a horizontal direction as viewed onthe page). In some embodiments, a width and spacing of the mask bars4-610, a thickness of the layer 4-622, and a thickness of the layer4-624 may be selected to produce approximately equivalent spacingbetween the sample wells in the second direction.

In some implementations, instead of a single material being depositedfor the planarizing layer 4-680 (as depicted in FIG. 4-6R), a stack ofmaterials may be deposited, for example, a combination of an insulator,a semiconductor, and a metal. The stack may then be planarized toproduce a layered structure for the sample well.

In FIG. 4-6S, the p- and n-type regions of the diodes are planar. Insome embodiments, and referring back to FIG. 4-6L, the exposed p- andn-type regions may be etched after patterning the resist 4-650 andbefore depositing the electrodes, as depicted in FIG. 4-6T. As anexample, a wet anisotropic etch may be used to selectively etch alongcrystallographic planes of the semiconductor. The etch may be a timedetch that undercuts the remaining resist 4-650 as indicated in thedrawing, according to some embodiments. The electrode material may thenbe deposited as depicted in FIG. 4-6U.

In some embodiments, the electrode material 4-520 may be a transparentconductor, for example ITO, so that emission from a sample may passthrough the electrode material. In some implementations, a thinsemiconductor layer 4-512 is used, so that absorption of emission from asample that passes through the semiconductor layer is less than about30%. For example, a thickness of the semiconductor layer from which thediodes are formed may be less than approximately 50 nm.

Any one or more of the foregoing embodiments of excitation sources maybe included in an embodiment of an integrated device.

V. Excitation Coupling

Coupling of energy from an excitation source to a sample well may beimproved or affected by forming excitation-coupling structures withinand/or adjacent a sample well. Excitation-coupling structures maycomprise micro- or nanoscale structures fabricated around a sample wellin some embodiments, or may comprise structures or particles formed at asample well in some embodiments. Excitation-coupling structures mayaffect radiative excitation of a sample in some implementations, and mayaffect non-radiative excitation of a sample in some implementations. Invarious embodiments, radiative excitation-coupling structures mayincrease an intensity of excitation energy within an excitation regionof a sample well. Non-radiative excitation-coupling structures mayimprove and/or alter non-radiative energy-transfer pathways from anexcitation source (which may be radiative or non-radiative) to a sample.

V. A. Radiative Plasmonic Excitation-Coupling Structures

There are a number of different types of radiative, excitation-couplingstructures that may be used to affect coupling of excitation energy froman excitation source to an excitation region within a sample well. Someradiative coupling structures may be formed of a conductor (e.g.,include a metal layer), and support surface plasmon oscillations thatlocally affect the excitation energy (e.g., locally alter anelectromagnetic field). In some cases, surface-plasmon structures mayenhance the excitation energy within an excitation region of the samplewell by a factor of two or more. Some radiative coupling structures mayalter the phase and/or amplitude of an excitation field to enhanceexcitation energy within a sample well. Various embodiments of radiativeexcitation-coupling structures are described in this section.

FIG. 5-1A depicts just one example of a surface-plasmon structure 5-120that may be used to enhance coupling of excitation energy into a samplewell. The drawing depicts a plan view of a region around asurface-plasmon structure 5-120, and represents results of a numericalsimulation of electric field intensity around the structure. The drawingdepicts a surface-plasmon structure comprising three triangular featureshaving sharp apexes that are located in close proximity to a sample well(not shown). According to some embodiments, a surface-plasmon structuremay comprise a metal or conductor (e.g., a patterned thin film of anyone or combination of the following metals or metal alloys: Al, Au, Ag,Ti, TiN). A thickness of the film may be between approximately 10 nm andapproximately 100 nm in some embodiments, though other thicknesses maybe used in other embodiments. A surface-plasmon structure, in someembodiments, may include sharp features 5-110 located in close proximityto a sample well (e.g., within about 100 nm).

FIG. 5-1B depicts a cross-section, elevation view of the surface-plasmonstructure of FIG. 5-1A, taken at the dashed line. The simulation shows alocalized, high-intensity region 3-505 of the excitation energy adjacentan apex of a triangle of the surface-plasmon structure. For thissimulation, the surface-plasmon structure 5-120 was located on adielectric layer 5-135 (e.g., silicon dioxide) above a waveguide 5-130.The surface-plasmon structure taps energy from an evanescent field ofthe waveguide, and enhances the intensity at the sample well.

In some embodiments, enhancement of excitation energy by asurface-plasmon structure may be localized to an extent that a samplewell 3-215 is not needed. For example, if a high-intensity region 3-505is formed having a diameter of approximately 100 nm with a peakintensity value greater than about 80% of the intensity outside theregion, then a deep sample well may not be needed. Only samples withinthe high-intensity region 3-505 may contribute appreciable emission forpurposes of detection.

When an incident electromagnetic field interacts with a surface-plasmonstructure, surface-wave currents are generated in the structure. Theshape of the structure can affect the intensity and distribution ofthese surface-plasmons. These localized currents can interact with andsignificantly alter and intensify the incident electromagnetic field inthe immediate vicinity of the surface-plasmon structure, e.g., asdepicted by the high-intensity region 3-505 in FIG. 5-1B. In someembodiments, an emitter (e.g., a fluorescing tag) that emits radiationnear a surface-plasmon structure can have its emission altered by thestructure, so as to alter a far-field radiation pattern from theemitter.

Another embodiment of a surface-plasmon structure 5-122 is depicted inthe plan view of FIG. 5-1C. The illustrated bow-tie structure comprisestwo triangular metallic structures located adjacent a sample well 3-210.The structures may be patterned below a sample well, for example, and/oradjacent an excitation region of the sample well. There may be a gap5-127 between the sample well and sharp features 5-125 of thesurface-plasmon structure, in some implementations. The gap 5-127 may bebetween approximately 10 nm and approximately 200 nm, according to someembodiments. In some implementations, the gap 5-127 may be betweenapproximately 10 nm and approximately 100 nm. The sharp features 5-125may comprise a point or sharp bend in an edge of the surface-plasmonstructure, as depicted in the drawing. The sharp features may have anysuitable shape. In some embodiments a bend radius of a sharp feature5-125 may be less than approximately five wavelengths associated withthe incident excitation energy. In some embodiments a bend radius of asharp feature 5-125 may be less than approximately two wavelengthsassociated with the incident excitation energy. In some embodiments abend radius of a sharp feature 5-125 may be less than approximately fivewavelengths associated with a surface-plasmon wave that is excited bythe incident excitation energy. In some embodiments a bend radius of asharp feature 5-125 may be less than approximately two wavelengthsassociated with a surface-plasmon wave that is excited by the incidentexcitation energy.

According to some embodiments, surface-plasmon structures 5-122 may bepatterned within a sample well 3-210 as illustrated in the elevationview of FIG. 5-1D. In some embodiments, a surface-plasmon structurewithin a sample well may comprise one or more fingers (e.g., metallicfingers) patterned onto sidewalls of the sample well, as depicted in thedrawing. FIG. 5-1E depicts a plan view of the sample well 3-210 showingthe surface-plasmon structures 5-122 formed on sidewalls within thesample well. In some embodiments, the lower ends of thesesurface-plasmon structures 5-122 form sharp features or bends where theelectromagnetic field will be enhanced. The surface-plasmon structures5-122 may, or may not, extend to a base of the sample well.

In some embodiments, the surface-plasmon structures 5-122 may bearranged to affect the polarization of the excitation energy and/oremitted radiation from the sample well. For example, a pattern asdepicted in FIG. 5-1E may be used to affect a preferred orientation oflinear or elliptical excitation polarization and/or a preferredorientation of linear or elliptical polarization from an emitter withinthe sample well.

Surface-plasmon structures may be patterned in shapes other than thosedepicted in FIG. 5-1A through FIG. 5-1E. For example, surface-plasmonstructures may be patterned as regular or periodic structures, asdepicted in FIG. 5-2A, according to some embodiments. For example, asurface-plasmon structure may be patterned is an array of protrudingfeatures 5-210 on a lower surface of a material 3-230 in which thesample well 3-210 is formed. Periodic surface-plasmon structures may beformed in a regular array, for example, a grating, a grid, a lattice, acircular grating, a spiral grating, an elliptical grating, or any othersuitable structure. In some implementations, there may be asubstantially uniform spacing s between the protrusions 5-210 of asurface-plasmon structure. In some implementations, the spacing s mayhave any value between approximately 40 nm and approximately 250 nm.According to some embodiments, the protrusions may have a height hbetween approximately 20 nm and approximately 100 nm. In someimplementations, the spacing s may be non-uniform or may be chirped(having a decreasing value at larger radial distances). In someembodiments, the protrusions 5-210 of a surface-plasmon structure may bepatterned as a Fresnel zone plate. According to some embodiments, asurface-plasmon structure of 5-210 may be formed adjacent a transparentlayer and/or dielectric layer 3-235.

In some implementations, a surface-plasmon structure 5-212 may be spacedfrom a material 3-230 in which the sample well is formed as depicted inFIG. 5-2B. For example, there may be an intervening dielectric layer5-247 between the surface-plasmon structure 5-212 and the material3-230. According to some embodiments, a surface plasmons structure 5-212may be located adjacent a divot 3-216 of a sample well, as depicted inthe drawing. For example, a surface-plasmon structure 5-212 may belocated adjacent sidewalls of a divot 3-216, as depicted in FIG. 5-2B.

FIG. 5-2C illustrates a surface-plasmon structure 5-214 that is formedas a concentric, circular grating. The structure 5-214 may compriseconcentric conducting rings 5-215, according to some embodiments. Therings may be separated by a regular spacing s and have a height h, asdescribed in connection with FIG. 5-2A. According to some embodiments, asample well 3-210 with an optional divot may be located at a center ofthe rings. The circular grating may be patterned adjacent a base of thesample well.

A periodicity of a surface-plasmon structure may be selected to form aresonant structure according to some embodiments. For example a spacings of a surface-plasmon structure may be selected to be approximatelyone-half wavelength of a surface-plasmon wave that is generated in thestructure by the excitation energy. When formed as a resonant structure,a surface-plasmon structure may accumulate and resonate excitationenergy along the direction of the periodic surface-plasmon structure.Such a resonant behaviour can intensify electromagnetic energy within asample well, or adjacent a sample well, as depicted in FIG. 5-2D.

FIG. 5-2D represents numerically simulated electromagnetic field resultsat the base of the sample well and around a periodic surface-plasmonstructure. The surface-plasmon structure 5-216 is located adjacent thematerial 3-230 in which the sample well is formed, and is adjacent abase of a sample well 3-210. The surface-plasmon structure may be in theform of a grating or circular grating that repeats at regular spacingintervals in regions away from the sample well and outside the simulatedregion. For example, there may be between three and 50 repeated gratingprotrusions of the surface-plasmon structure 5-216. A region of highintensity 5-240 can be seen at the base of the sample well 3-210. Theintensity within this region has been enhanced by more than a factor of2 over the surrounding region just below the surface-plasmon structure.

FIG. 5-2E depicts, in elevation view, an alternative embodiment of aresonant surface-plasmon structure 5-218. According to some embodiments,a surface-plasmon structure may be formed as periodic grating or gridpatterns, and may be patterned in multiple layers 5-247. A sample well3-210 may be patterned through the multiple layers 5-247 and within theresonant surface-plasmon structure 5-218, according to some embodiments.In some implementations, a resonant surface-plasmon structure maycomprise discrete conductive elements 5-222 is depicted in the plan viewof FIG. 5-2F. In some implementations, a resonant surface-plasmonstructure may comprise a continuous lattice pattern 5-250, as depictedin FIG. 5-2G. A dielectric filler 5-252 may be located in voids of theconductive material 5-250, and a sample well 3-210 may be located with avoid.

There are a variety of different surface-plasmon structures that may beused to enhance coupling into a sample well or to affect emission from asample within the sample well. FIG. 5-2H depicts, in plan view, yet analternative embodiment of the surface-plasmon structure. An elevationview of the structure is depicted in FIG. 5-2I. According to someimplementations, a surface-plasmon structure may comprise an array ofdiscs distributed around a sample well 3-210. In some implementations,instead of using conductive discs 5-260, a surface-plasmon structure maycomprise a conductive layer through which a distributed pattern of holesis formed. Such a structure may be referred to as a “nano-antenna.”

V. B. Fabrication of Plasmonic Excitation-Coupling Structures

A variety of different processes may be used to pattern surface-plasmonstructures adjacent a sample well. FIG. 5-3A through FIG. 5-5E depictstructures associated with process steps that may be used to formsurface-plasmon structures adjacent to a sample well, according to someembodiments. Referring now to FIG. 5-3A, a process for forming asurface-plasmon structure may comprise forming a resist layer 5-310 onan anti-reflective coating (ARC) 5-320 on a masking layer 5-330. Thelayers may be disposed on a transparent dielectric layer 3-235,according to some implementations. The resist layer 5-310 may comprise aphotoresist or an electron- or ion-beam resist that may belithographically patterned. The masking layer 5-330 may comprise a hardmask formed of an inorganic material (e.g., silicon or silica nitride,or any other suitable material), according to some embodiments.

In some implementations, a photolithographic process may be used topattern the resist 5-310 as depicted in FIG. 5-3B. The selected patternmay comprise a layout of protrusions or holes that will be used to forma desired surface-plasmon structure. After development of the resist5-310, regions of the ARC will be exposed, and the pattern may be etchedinto the ARC layer 5-320 and then into the masking layer 5-330. Theresist and ARC may be stripped from the substrate, and a resultingstructure may appear as shown in FIG. 5-3C. The masking layer 5-330 maythen be used as an etch mask, so that the pattern may be transferredinto the underlying dielectric layer 3-235 via a selective anisotropicetch, as depicted in FIG. 5-3D.

A conductive material 3-230, or a layer of materials comprising aconductor, may then be deposited over the region, as illustrated in FIG.5-3E. Any suitable conductive material may be used for forming a surfaceplasmon structure, whether or not it is deposited as a separate layerfrom the material 3-230. For example, in some cases, a first conductivematerial may be deposited as a base layer of material 3-230 in which asurface-plasmon structure is formed. Examples of materials that may beused for forming a surface-plasmon structure include, but are notlimited to, Au, Al, Ti, TiN, Ag, Cu, and alloys or combination layersthereof.

The material 3-230, or layer of materials, may be deposited by anysuitable deposition process, including but not limited to a physicaldeposition process or a chemical vapor deposition process. In someembodiments, the material 3-230 may have a thickness betweenapproximately 80 nm and approximately 300 nm. In some implementations,the material 3-230 may be planarized (e.g., using a CMP process), thoughplanarization is not necessary. A sample well may be formed in thematerial 3-230 using any suitable process described herein in connectionwith fabricating a sample well.

The inventors have recognized that forming a surface-plasmon structureaccording to the steps shown in FIG. 5-3A through FIG. 5-3E may requireaccurate alignment of the sample well to the surface-plasmon structure.For example, a surface-plasmon structure comprising a concentricgrating, as depicted in FIG. 5-2C, may require accurate alignment of thesample well 3-210 to the center of the surface-plasmon structure 5-214.To avoid fabrication difficulties associated with such accuratealignment, the inventors have developed self-alignment processes thatare depicted in FIG. 5-4A through FIG. 5-5E.

Referring now to FIG. 5-4A, a process for forming a surface-plasmonstructure and sample well that is self-aligned to the surface-plasmonstructure may comprise forming a masking layer 5-410 on a transparentdielectric layer 3-235. The masking layer may comprise a hard maskformed of an inorganic material, such as silicon or silica nitride,according to some embodiments. A thickness of the masking layer 5-410may be approximately equal to a desired height of a sample well 3-210.For example, the thickness of the masking layer may be betweenapproximately 50 nm and approximately 200 nm, according to someembodiments, though other thicknesses may be used in other embodiments.

The masking layer 5-410 may be patterned to create voids 5-430 havingthe desired pattern of a surface-plasmon structure that will bepatterned in the dielectric layer 3-235. The patterning of the maskinglayer 5-410 may be done with any suitable lithography process (e.g.,photolithography, electron-beam lithography, ion-beam lithography, EUVlithography, x-ray lithography). The resulting structure may appear asshown in FIG. 5-4B. The structure may include a central pillar 5-420,which will be used subsequently to form the self-aligned sample well.

A resist 5-440 (e.g., a photoresist) may then be patterned over thepatterned masking layer 5-410, as depicted in FIG. 5-4C. Alignment forpatterning the resist 5-440 (e.g., mask to substrate alignment) need notbe highly accurate, and only requires the resist 5-440 to cover acentral pillar 5-420 and not cover voids 5-430 that will be used to formthe surface-plasmon structure.

A selective anisotropic etch may then be used to etch the dielectriclayer 3-235 and transfer the pattern of the surface-plasmon structureinto the dielectric, as depicted in FIG. 5-4D according to someembodiments. A selective isotropic etch may then be used to remove theexposed portions of the masking layer 5-410. The isotropic etch may be awet etch, for example, though an isotropic dry etch may be used in someembodiments. Because the resist 5-440 covers the central pillar 5-420,the central pillar will not be etched and remain on the substrate, asdepicted in FIG. 5-4E. The resist 5-440 may then be stripped from thesubstrate exposing the pillar 5-420, as depicted in FIG. 5-4F.

According to some embodiments, a metal conductive material 3-230, or astack of materials including a conductive material, may then bedeposited over the region as illustrated in FIG. 5-4G. The centralpillar 5-420 and a cap of deposited material over the pillar may then beremoved by a selective wet etch of the pillar, lifting off the cap. Theremoval of the central pillar leaves a sample well that is self-alignedto the underlying surface-plasmon structure 5-450.

An alternative process may be used to form a sample well that isself-aligned to a surface-plasmon structure, and is depicted in FIG.5-5A through FIG. 5-5E. According to some embodiments, one or moreconductive layers 5-510, 5-520 may be patterned on a transparentdielectric layer 3-235 using any suitable lithography process, asdepicted in FIG. 5-5A. In some implementations, a first layer 5-510 maycomprise aluminum, and a second layer 5-520 may comprise titaniumnitride, though other material combinations may be used in variousembodiments. A total thickness of the one or more layers may beapproximately equivalent to a desired height of the sample well,according to some embodiments. The patterning may form a sample well3-210, and voids 5-525 adjacent the sample well in the one or more metallayers. The voids may be arranged in the pattern of a desiredsurface-plasmon structure.

In some implementations, the dielectric layer 3-235 may be etched totransfer the pattern of the surface-plasmon structure and sample well3-210 into the dielectric layer, as depicted in FIG. 5-5B. The etchdepth into the dielectric may be between approximately 20 nm andapproximately 150 nm, according to some embodiments. A resist 5-440 maybe patterned to cover the sample well, as depicted in FIG. 5-5C.Alignment for patterning the resist need not be highly accurate, andonly need cover the sample well without covering adjacent etched regionsof the dielectric layer 3-235 that will be used to form thesurface-plasmon structure.

As illustrated in FIG. 5-5D, a conductive material 5-512, or layers ofmaterials including a conductor, may be deposited over the region usingany suitable deposition process. The material 5-512 may fill the etchedregions of the dielectric layer, and may extend above the one or morelayers 5-510, 5-520. The resist 5-440 and the material covering theresist may then be removed according to a lift-off process. Theresulting structure, shown in FIG. 5-5E, leaves a sample well that isself-aligned to the surrounding surface-plasmon structure. The samplewell includes a divot 3-216.

In some embodiments the process depicted in FIG. 5-5A through FIG. 5-5Emay be used to form a sample well that does not have a divot 3-216. Forexample, the resist 5-440 may be patterned over the sample well 3-210before the dielectric layer 3-235 is etched. The dielectric layer 3-235may then be etched, which will transfer the pattern of thesurface-plasmon structure to the dielectric layer but not form a divot.The process may then proceed as illustrated in FIG. 5-5D and FIG. 5-5 Eto create a self-aligned sample well having no divot.

V. C. Amplitude/Phase Excitation-Coupling Structures

Other structures, in addition to or as an alternative to surface-plasmonstructures, may be patterned in the vicinity of the sample well 3-210 toincrease the excitation energy within the sample well. For example somestructures may alter the phase and/or the amplitude of the incidentexcitation field so as to increase the intensity of the excitationenergy within the sample well. FIG. 5-6A depicts a thin lossy film 5-610that may be used to alter the phase and amplitude of incident excitationradiation and increase the intensity of electromagnetic radiation withinthe sample well.

According to some embodiments, a thin lossy film may create constructiveinterference of the excitation radiation, resulting in field enhancementwithin an excitation region of the sample well. FIG. 5-6B depicts anumerical simulation of excitation radiation incident upon a sample wellwhere a thin lossy film 5-610 has been formed immediately adjacent thesample well. For the simulation, the sample well has a diameter ofapproximately 80 nm and is formed in a metallic layer of goldapproximately 200 nm thick. The sample well comprises an SCN, andsuppresses propagation of excitation radiation through the sample well.The thin lossy film 5-610 is approximately 10 nm thick, is formed fromgermanium, and covers an underlying transparent dielectric comprisingsilicon dioxide. The thin lossy film extends across an entrance apertureof the sample well. The simulation shows that the intensity of theexcitation radiation is a highest value at the entrance aperture of thesample well. The intensity of the excitation radiation in this brightregion 5-620 is more than twice the value of the intensity to the leftand right of the sample well.

A thin lossy film may be made from any suitable material. For example, athin lossy film may be made from a material where the index ofrefraction n is approximately the same order of magnitude as theextinction coefficient k for the material. In some embodiments, a thinlossy film may be made from a material where the index of refraction nis within about two orders of magnitude difference from the value of theextinction coefficient k of the material. Non-limiting examples of suchmaterials at visible wavelengths are germanium and silicon.

A thin lossy film may be any suitable thickness, which may depend upon acharacteristic wavelength, or wavelengths, associated with theexcitation source, or sources. In some embodiments, a thin lossy filmmay be between approximately 1 nm and approximately 45 nm thick. Inother embodiments, a thin lossy film may be between approximately 15 nmand approximately 45 nm thick. In still other embodiments, a thin lossyfilm may be between approximately 1 nm and approximately 20 nm thick.

Effects of a thin lossy film on reflectance from the material 3-230 inwhich a sample well is formed, excitation energy loss within the thinlossy film, and excitation energy loss within the material 3-230 areshown in the graph of FIG. 5-6C. One curve plotted in the graphrepresents a reflectance curve 5-634, and shows how reflectance from thematerial 3-230 and the thin lossy film 5-610 vary as the thickness ofthe thin lossy film changes from 0 nm to 100 nm. The reflectance reachesa minimum value at about 25 nm, according to the simulated embodiment.The reflectance minimum will occur at different thicknesses depending ona characteristic wavelength of the excitation energy and materials usedfor the thin lossy film and material 3-230. In some implementations athickness of thin lossy film is selected such that the reflectance isapproximately at its minimal value.

In some embodiments, a thin lossy film 5-610 may be spaced from a samplewell 3-210 and material 3-230, as depicted in FIG. 5-6D. For example, athin dielectric layer 5-620 (e.g., a silicon oxide SiO_(x)) may beformed over a thin lossy film, and a sample well 3-210 may be formedadjacent the dielectric layer 5-620. A thickness of the dielectric layer5-620 may be between approximately 10 nm and approximately 150 nmaccording to some embodiments, though other thicknesses may be used insome embodiments.

Although depicted as a single layer, a thin lossy film may comprisemultiple layers of two or more materials. In some implementations, amultilayer stack comprising alternating layers of a thin lossy film5-610 and a dielectric layer 5-620 may be formed adjacent a sample well3-210, as depicted in FIG. 5-6E. A thickness of a thin lossy film 5-610in a stack of layers may be between approximately 5 nm and approximately100 nm, and a thickness of a dielectric layer 5-620 within the stack maybe between approximately 5 nm and approximately 100 nm, according tosome embodiments. In some implementations, the multilayer stack maycomprise a layer of silicon dioxide having a thickness betweenapproximately 2 nm and approximately 8 nm, a layer of silicon having athickness between approximately 5 nm and approximately 20 nm, and alayer of germanium having a thickness between approximately 2 nm andapproximately 12 nm, though other thicknesses may be used in otherembodiments. In some implementations, the multilayer stack may comprisea layer of silicon dioxide (approximately 4.2 nm thick), a layer ofsilicon (approximately 14.4 nm thick), and a layer of germanium(approximately 6.5 nm thick), though other thicknesses may be used inother embodiments.

A thin lossy film may be fabricated from any suitable material thatexhibits at least some loss to the incident radiation. In someembodiments, a thin lossy film may comprise a semiconductor material,for example silicon and germanium, though other materials may be used(e.g., SiGe, Ga, N, C, GaN, InP, AlGaN, InGaP, etc.). In someimplementations, a thin lossy film may comprise inorganic material or ametal. In some embodiments, a thin lossy film may comprise an alloy orcompound semiconductor. For example, a thin lossy film may comprise analloy including Si (57.4% by weight), Ge (25.8% by weight), and SiO₂(16.8% by weight), though other ratios and compositions may be used inother embodiments.

According to some embodiments, a thin lossy film may be formed on thesubstrate using any suitable blanket deposition process, for example, aphysical deposition process, a chemical vapor deposition process, a spinon process, or a combination thereof. In some embodiments, a thin lossyfilm may be treated after deposition, e.g., baked, annealed and/orsubjected to ion implantation.

Other phase/amplitude altering structures may be used additionally oralternatively to enhance excitation energy within the sample well.According to some implementations and as shown in FIG. 5-7A, areflective stack 5-705 may be spaced from a sample well 3-210. In someembodiments, a reflective stack may comprise a dielectric stack ofmaterials having alternating indices of refraction. For example a firstdielectric layer 5-710 may have a first index of refraction, and asecond dielectric layer 5-720 may have a second index of refractiondifferent than the first index of refraction. The reflective stack 5-705may exhibit a high reflectivity for excitation radiation in someembodiments, and exhibit a low reflectivity for radiative emission froman emitter within the sample well. For example, a reflective stack 5-705may exhibit a reflectivity greater than approximately 80% for excitationradiation and a reflectivity lower than approximately 40% for emissionfrom a sample, though other reflectivity values may be used in someembodiments. A dielectric layer 5-730 that transmits the excitationenergy may be located between the reflective stack and the sample well.

According to some implementations, a reflective stack 5-705 depicted inFIG. 5-7A may form a resonator or resonant cavity with the material3-230 in which the sample well 3-210 is formed. For example, thereflective stack may be spaced from the material 3-230 by a distancethat is approximately equal to one-half the wavelength of the excitationradiation within the dielectric material 5-730, or an integral multiplethereof. By forming a resonator, excitation energy may pass through thereflective stack, resonate, and build up in the space between thematerial 3-230 and the reflective stack 5-705. This can increaseexcitation intensity within the sample well 3-210. For example, theintensity may increase within the resonant structure by more than afactor of 2 in some embodiments, and more than a factor of 5 in someembodiments, and yet more than a factor of 10 in some embodiments.

A resonant cavity formed at the sample well may comprise aGires-Tournois resonator, according to some embodiments. In someimplementations, a resonant structure may comprise a linear resonantcavity or ring resonator. In some implementations, a resonant structuremay comprise a distributed Bragg reflector formed adjacent the samplewell. The distributed Bragg reflector may comprise alternating layers ofmaterial having different indices of refraction. In someimplementations, a resonant cavity may comprise a microcavity. Themicrocavity may have microscale dimensions. In some aspects, amicrocavity may have a size that is approximately equal to one-half thecharacteristic wavelength of an excitation source or an integralmultiple thereof (as modified by the refractive index n of the resonantcavity). For example, the dimension of a microcavity may be Mλ/2n, whereM is an integer.

Additional structures may be added in the vicinity of the sample well,as depicted in FIG. 5-7B and FIG. 5-7C. According to some embodiments, adielectric plug 5-740 having a first index of refraction that is higherthan a second index of refraction of the dielectric layer 5-730 may beformed adjacent the sample well 3-210, as depicted in FIG. 5-7B. Theplug may be in the shape of a cylinder having a diameter approximatelyequal to that of the sample well, though other shapes and sizes may beused. Because of its higher refractive index, the dielectric plug 5-740may condense and guide excitation radiation toward the sample well.

A dielectric structure, such as the plug 5-740, may be used with orwithout a reflective stack 5-705, according to some embodiments. Such adielectric structure may be referred to as a dielectric resonantantenna. The dielectric resonant antenna may have any suitable shape,for example, cylindrical, rectangular, square, polygon old, trapezoidal,or pyramid.

FIG. 5-7C and FIG. 5-7D depict a photonic bandgap (PBG) structure thatmay be formed in the vicinity of a sample well 3-210, according to someembodiments. A photonic bandgap structure may comprise a regular arrayor lattice of optical contrast structures 5-750. The optical contraststructures may comprise dielectric material having a refractive indexthat is different from a refractive index of the surrounding dielectricmaterial, according to some embodiments. In some implementations, theoptical contrast structures 5-750 may have a loss value that isdifferent from the surrounding medium. In some implementations, a samplewell 3-210 may be located at a defect in the lattice as depicted in FIG.5-7D. According to various embodiments, the defect in the photoniclattice may confine photons within the region of the defect can enhancethe intensity of the excitation energy at the sample well. Theconfinement due to the photonic bandgap structure may be substantiallyin two dimensions transverse to a surface of the substrate. Whencombined with the reflective stack 5-705, confinement may be in threedimensions at the sample well. In some embodiments, a photonic bandgapstructure may be used without a reflective stack.

Various methods have been contemplated for fabricating theexcitation-coupling structures depicted in FIG. 5-6A through FIG. 5-7D.Structures that require thin planar films (e.g., dielectric films ofalternating refractive index) may be formed by planar depositionprocesses, according to some embodiments. Planar deposition processesmay comprise physical deposition (for example, electron beam evaporationor sputtering) or chemical vapor deposition processes. Structures thatrequire discrete embedded dielectrics formed in three-dimensionalshapes, such as a dielectric resonant antenna 5-740 shown in FIG. 5-7Bor the optical contrast structures 5-750 shown in FIG. 5-7C, may beformed using lithographic patterning and etching processes to etch thepattern into the substrate, and using subsequent deposition of adielectric layer, and a planarization of the substrate, for example Alsocontemplated are self-alignment processing techniques for formingdielectric resonant antennas as well as photonic bandgap structures inthe vicinity of the sample well 3-210.

V. D. Fabrication of Amplitude/Phase Excitation-Coupling Structures

FIG. 5-8A through FIG. 5-8G depict structures associated with processsteps for just one self-alignment process that may be used to form aphotonic bandgap structure and a self-aligned sample well as illustratedin FIG. 5-7C. According to some embodiments, a reflective stack 5-705may be first formed on a substrate above a dielectric layer 3-235, asillustrated in FIG. 5-8A. A second dielectric layer 5-730 may then bedeposited over the reflective stack. The thickness of the dielectriclayer 5-730 may be approximately equal to about one-half a wavelength ofthe excitation radiation in the material, or an integral multiplethereof. Process steps described in connection with FIG. 5-4A throughFIG. 5-4E may then be carried out to form a pillar 5-420 above thedielectric layer 5-730 and a pattern of etched features 5-810 for thephotonic bandgap structure. The etched features may extend into thedielectric layer 5-730 and optionally into the reflective stack 5-705.The resulting structure may appear as shown in FIG. 5-8A.

A resist 5-440 covering the pillar 5-420 may be stripped from thesubstrate and a conformal deposition performed to fill the etchedfeatures with a filling material 5-820, as depicted in FIG. 5-8B. Thefilling material 5-820 may be the same material that is used to form thepillar 5-420, according to some embodiments. For example the fillingmaterial 5-820 and the pillar 5-420 may be formed of silicon nitride andthe dielectric layer 5-730 may comprise an oxide, e.g., SiO₂.

An anisotropic etch may then be carried out to etch back the fillingmaterial 5-820. The filling material may be etched back to expose asurface of the dielectric layer 5-730, according to some embodiments,resulting in a structure as depicted in FIG. 5-8C. The etch may leave apillar 5-830 comprising the original pillar 5-420 and sidewalls 5-822that remain from the filling material 5-820.

A resist 5-440 may then be patterned over the substrate as depicted inFIG. 5-8D. For example, the resist may be coated onto the substrate, ahole patterned in the resist, and the resist developed to open up aregion in the resist around the pillar 5-830. Alignment of the hole tothe pillar need not be highly accurate, and only need expose the pillar5-830 without exposing the underlying photonic bandgap structuresembedded in the dielectric layer 5-730.

After the pillar 5-830 is exposed, and isotropic etch may be used toreduce the transverse dimension of the pillar. According to someembodiments, the resulting pillar shape may appear as depicted in FIG.5-8E. The resist 5-440 may then be stripped from the substrate and amaterial 3-230, or layers of materials, may be deposited over theregion. In some embodiments, the material 3-230 may be etched back usinga CMP process to planarize the region as depicted in FIG. 5-8F.Subsequently, a selective dry wet etch may be used to remove theremaining pillar structure leaving a sample well 3-210, as illustratedin FIG. 5-8G. As indicated by the drawings, the sample well 3-210 isself-aligned to the photonic bandgap structure patterned in thedielectric layer 5-730.

As an alternative process, the filling material 5-820 may comprise adifferent material than the material used to form the pillar 5-420. Inthis process, the steps associated with FIG. 5-8D and FIG. 5-8E may beomitted. After deposition of material 3-230 and planarization, asdepicted in FIG. 5-8F, a selective etch may be performed to remove thepillar 5-420. This may leave sidewalls of the filling material 5-820lining the sample well 3-210.

V. E. Non-Radiative Excitation-Coupling Structures and Fabrication

Structures for non-radiative coupling of excitation energy to a samplewithin the sample well have also been contemplated by the inventors.Just one embodiment of a non-radiative coupling structure is depicted inFIG. 5-9A. According to some embodiments, a non-radiative couplingstructure may comprise a semiconductor layer 5-910 formed immediatelyadjacent a sample well 3-210. The semiconductor layer 5-910 may be anorganic semiconductor in some embodiments, or an inorganic semiconductorin some embodiments. In some implementations, a divot 3-216 may, or maynot, be formed in the semiconductor layer. The semiconductor layer 5-910may have a thickness between approximately 5 nm and approximately 100 nmaccording to some embodiments, though other thicknesses may be used insome embodiments. According to some implementations, excitationradiation or photons 5-930 from an excitation source may impinge uponthe semiconductor layer 5-910 and produce excitons 5-920. The excitonsmay diffuse to a surface of the sample well where they maynon-radiatively recombine and transfer energy to a sample adjacent thewalls of the sample well.

FIG. 5-9B depicts another embodiment in which a semiconductor layer5-912 may be used to non-radiatively transfer energy from excitationenergy to a sample. In some embodiments, a semiconductor layer 5-912 maybe formed at the bottom of a sample well or in a divot of the samplewell 3-210, as depicted in the drawing. The semiconductor layer 5-912may be formed in a sample well by using a directional deposition processas described herein in connection with process steps for depositing anadherent at the base of the sample well, according to some embodiments.The semiconductor layer 5-912 may have a thickness between approximately5 nm and approximately 100 nm according to some embodiments, thoughother thicknesses may be used in other embodiments. Incident radiationmay generate excitons within the semiconductor layer, which may thendiffuse to the a bottom surface of the sample well 3-210. The excitonsmay then non-radiatively transfer energy to a sample within the samplewell.

Multiple non-radiative pathways for transferring excitation energy to asample have also been contemplated by the inventors. According to someembodiments, and as depicted in FIG. 5-9C, an energy-transfer particle5-940 may be deposited within a sample well. The energy-transferparticle may comprise a quantum dot in some embodiments, or may comprisea molecule in some embodiments. In some implementations, theenergy-transfer particle 5-940 may be functionalized to a surface of thesample well through a linking molecule. A thin semiconductor layer 5-910may be formed adjacent the sample well, or within the sample well, andexcitons may be generated within the semiconductor layer from theexcitation radiation incident upon the semiconductor layer, as depictedin the drawing. The excitons may diffuse to the surface of the samplewell, and non-radiatively transfer energy to the energy-transferparticle 5-940. The energy-transfer particle 5-940 may thennon-radiatively transfer energy to a sample 3-101 within the samplewell.

According to some implementations, there may be more than oneenergy-transfer particle 5-940 within a sample well. For example, alayer of energy-transfer particles 5-942 may be deposited within asample well, such as the sample well depicted in FIG. 5-9C.

FIG. 5-9D illustrates a layer of energy-transfer particles 5-942deposited at a base of a sample well adjacent to an electrically-excitedsemiconductor layer 4-510. Excitons generated within the semiconductorlayer 4-510 may transfer energy non-radiatively to the energy-transferparticles 5-942 which may in turn transfer energy non-radiatively to asample 3-101 within the well. A structure depicted in FIG. 5-9D isdescribed herein in connection with FIG. 4-5A.

In some implementations, energy-transfer particles 5-942, or a singleenergy-transfer particle 5-940, may be deposited at a base of a samplewell, as depicted in FIG. 5-9E. The energy-transfer particle, orparticles, may radiatively or non-radiatively transfer excitation energyto a sample 3-101 within the well. For example, an energy-transferparticle may absorb incident radiation to form an excited state of theenergy-transfer particle, and then radiatively or non-radiativelytransfer energy to the sample 3-101.

In some implementations, an energy-transfer particle may absorb incidentexcitation energy, and then re-emit radiative energy at a wavelengththat is different than the wavelength of the absorbed excitation energy.The re-emitted energy may then be used to excite a sample within thesample well. FIG. 5-9F represents spectral graphs associated with adown-converting energy-transfer particle. According to some embodiments,a down-converting energy-transfer particle comprises a quantum dot thatmay absorb short wavelength radiation (higher energy), and emit one ormore longer wavelength radiations (lower energy). An example absorptioncurve 5-952 is depicted in the graph as a dashed line for a quantum dothaving a radius between 6 to 7 nm. The quantum dot may emit a first bandof radiation illustrated by the curve 5-954, a second band of radiationillustrated by the curve 5-956, and a third band of radiationillustrated by the curve 5-958.

In some implementations an energy-transfer particle may up convertenergy from an excitation source. FIG. 5-9G depicts spectra associatedwith up conversion from an energy-transfer particle. According to someembodiments, a quantum dot may be excited with radiation atapproximately 980 nm, and then re-emit into one of three spectral bandsas illustrated in the graph. A first band may be centered atapproximately 483 nm, a second band may be centered at approximately 538nm, and a third band may be centered at approximately 642 nm. There-emitted photons from the quantum dot are more energetic than thephotons of the radiation used to excite the quantum dot. Accordingly,energy from the excitation source is up-converted. One or more of theemitted spectral bands may be used to excite one or more one or moresamples within the sample well.

Any one or more of the foregoing embodiments of excitation-couplingstructures may be included in an embodiment of an integrated device.

VI. Emission Coupling

One or more emission-coupling components may be formed between a samplewell and a corresponding sensor in a pixel to improve collection ofemission energy from the sample well by the sensor. Emission-couplingcomponents may improve the signal-to-noise ratio of the emission energysignal to a background signal in order to improve detection of a tag,for example, for purposes of analyzing a sample. According to someembodiments, emission-coupling components may be configurationed tospatially direct and/or spatially separate emission energies ofdifferent characteristic wavelengths.

In some implementations, emission-coupling components may directexcitation energy from a sample well to one or more sensor segmentswithin a pixel. In some embodiments, the location of theemission-coupling structure with respect to the sample well is selectedso as to direct the emission energy from the sample well in a particularway toward one or more sensor segments. For example, anemission-coupling structure may be configured to direct emission energyinto a radiation distribution pattern which has a shape that depends onthe characteristic wavelength emitted by a tag. A sensor may beconfigured to discern different spatial distribution patterns andproduce signals that can be analyzed to differentiate between thedifferent patterns. Accordingly, multiple different tags, each emittingwithin different spectral bands, may be distinguishable by theirrespective radiation patterns that form when the emission couples to,and is affected by, an emission-coupling structure formed adjacent thesample well. Other components, such as filters, may be included within apixel and may reduce background radiation (e.g., excitation energy andother energy not associated with emission from the sample).

VI. A. Surface Optics

Emission-coupling components or structures may be formed within a pixeland located near the sample well (e.g., within about 5 microns from thesample well in some implementations). These emission-coupling componentsmay be referred to as “surface optics,” and may support surfaceplasmons. In various embodiments, emission-coupling components may beconfigured to couple with and affect or alter the emission from a samplewith the sample well. In some embodiments, surface-optical structuresmay be formed at an interface between two layers within a pixel of theintegrated device. For example, some emission-coupling components may beformed at the interface between a layer in which the sample well isformed and an adjacent layer at an entrance aperture end of the samplewell. In some instances, the layer adjacent the sample well is adielectric layer, though other materials (e.g., lossy films,semiconductor, transparent conductor) may be used for the adjacentlayer.

Surface-energy coupling elements may be surface optical structures thatare excited by and interact with radiative emission from the samplewell. The surface optical structures may be configured to form differentspatial radiation patterns for emission energies of differentcharacteristic wavelengths.

A characteristic dimension of a surface-optical structure, such as agrating period, feature size, or distance from the sample well may beselected to maximally couple a parallel component of an emission energymomentum vector into a surface wave momentum vector for a surfaceplasmon. For example, the parallel component of the emission energymomentum vector may be matched to the surface wave momentum vector for asurface plasmon supported by the structure, according to someembodiments. In some embodiments, a distance d from the sample well toan edge or characteristic feature of a surface optical structure may beselected so as to direct emission energy from the sample well in aselected direction, such as normal to the surface or inclined at anangle θ from normal to the surface. For example, the distance, d, may bean integral number of surface-plasmon wavelengths for directing emissionnormal to the surface. In some embodiments, distance, d, may be selectedto be a fractional surface-plasmon wavelength, or wavelength modulothereof, for directing emission at an angle θ from normal to thesurface.

In operation, an emission-coupling component and sample well may beconfigured to increase the amount of emission energy that is radiatedfrom the sample well toward one or more sensor segments in the pixelcontaining the sample well. Without an emission-coupling component, anexcited sample may emit radiation in a half-shell or Lambertiandistribution due mainly to the presence of the layer in which the samplewell is formed. If the sample well allows propagation of emissionenergy, some emission may go into the bulk specimen. If the sample wellcomprises a ZMW, for example, the emission may be approximatelyLambertian in the direction of a sensor. The addition ofemission-coupling components at the sample well may create a highlyanisotropic emission distributions that may significantly differ from aLambertian distribution, and the distribution pattern may be dependentupon emission wavelength.

According to some embodiments, an emission-coupling structure may coupleradiative emission energy from a sample well at a first characteristicwavelength in a first direction and/or in a first characteristic spatialpattern. The coupled energy may be directed in the first direction in anarrowed, anisotropic radiation pattern, for example. In someembodiments, the emission-coupling structure may further coupleradiative emission energy from the same sample well at a secondcharacteristic wavelength in a second direction and/or secondcharacteristic spatial pattern that is different from the firstdirection and/or in a first characteristic spatial pattern. The secondemission may also be direction in a narrowed, anisotropic radiationpattern. In some embodiments, radiation with a first characteristicwavelength is directed in a narrowed lobe normal to the surface at whichthe surface optical structure is formed, and radiation of a secondcharacteristic wavelength is directed in annular lobes at an angle fromnormal to the surface. Other spatial distribution patterns may beproduced at other characteristic wavelengths for the sameemission-coupling structure.

A non-limiting example of an emission-coupling structure is a concentricgrating, as depicted in FIG. 6-1A. According to some embodiments, aconcentric grating structure may be formed in a pixel of the integrateddevice and configured to direct emission energy towards one or moresensor segments within the pixel. A concentric grating may compriseannular rings or protrusions, arranged in a bulls-eye pattern, formedaround a sample well. The concentric grating structure may couple withemission from the sample well to improve propagation of emission energyout of the sample well and concentration of the emission energy at oneor more sensor segments within the pixel.

An example of a concentric, circular grating 6-102 emission-couplingstructure is depicted in FIG. 6-1A. The circular grating may compriseany suitable number of rings and the number of rings shown in FIG. 6-1Ais a non-limiting example. The circular grating may comprise protrudingrings from a surface of a conductive film. For example, the circulargrating may be formed at the interface of the sample well layer and adielectric layer formed underneath the sample well layer. The samplewell layer may be a conductive material and the concentric grating maybe formed by patterning the grating structure at the interface betweenthe conductive material and the dielectric. The rings of the circulargrating may be on a regular periodic spacing, or may have irregular oraperiodic spacings between the rings. The sample well may be located ator near the center of the circular grating. In some embodiments, thesample well may be located off-center to the circular grating and may bepositioned a certain distance from the center of the grating.

In some embodiments, a grating-type emission-coupling component maycomprise a spiral grating. An example of a spiral grating 6-202 isdepicted in FIG. 6-1B. A spiral grating 6-202 may comprise a spiralaperture in a conductive film in some embodiments, or may comprise aspiral protrusion formed on a conductive layer according to someembodiments. Any suitable dimensions of the spiral grating may be usedto form the spiral grating.

A grating structure such as those depicted in FIG. 6-1A or FIG. 6-1Bformed adjacent a sample well may produce different spatial distributionpatterns for emission originating from the sample well. Examples ofpossible spatial distribution patterns that may form due to theinfluence of the grating is depicted in FIG. 6-2A through FIG. 6-2D. Forexample, a layer 6-306 of an integrated device may contain a sample wellwith a concentric grating structure 6-302 positioned underneath thesample well. When emission energy having a first characteristicwavelength is emitted by a sample in the sample well, the emissionenergy couples with the concentric grating and forms a first spatialdistribution pattern 6-304 illustrated in FIG. 6-2A. Additionally, whenemission energy having a second characteristic wavelength is emitted bya sample in the sample well, a second distribution pattern may form,such as the distribution pattern 6-404 shown in FIG. 6-2B. Similarly,FIG. 6-2C illustrates a third spatial distribution pattern 6-504 foremission energy having a third characteristic wavelength and FIG. 6-2Dillustrates a fourth spatial distribution pattern 6-604 having a fourthcharacteristic wavelength. The different spatial distribution patternsmay be detected by spatially separated sensor segments within the pixelto differentiate among the first, second, third, and fourthcharacteristic wavelengths.

Another example of a surface optic or surface plasmon structure is anano-antenna structure, an example of which is depicted in FIG. 6-3A. Anano-antenna structure may be configurationed to spatially direct and/orspatially separate emission energies of different characteristicwavelengths. In some embodiments, the location of the nano-antennastructure with respect to the sample well is selected so as to directthe emission energy from the sample well in a particular way toward oneor more sensor segments. Nano-antennas may comprise nanoscale dipoleantenna structures that are configurationed to produce a directionalradiation pattern when excited by emission energy. The nano-antennas maybe distributed around a sample well. The directional radiation patternmay result from a summation of the antennas' electromagnetic fields. Insome embodiments, the directional radiation pattern may result from asummation of the antennas' electromagnetic fields with the field emitteddirectly from the sample. In some implementations, the field emitteddirectly from the sample may be mediated by surface plasmon wavesassociated with the nano-antenna structure.

The dimensions of the individual nano-antennas that form thenano-antenna structure may be selected for the combined ability of theoverall nano-antenna structure to produce specific distribution patternsof one or more emission energies. For example, the diameters of theindividual nano-antennas may vary within a nano-antenna structure.However, in some instances, the diameters may be the same within a setof nano-antennas. In other implementations, a few selected diameters maybe used throughout the overall nano-antenna structure. Somenano-antennas may be distributed on a circle of radius R and some may beshifted in a radial direction from the circle. Some nano-antennas may beequally spaced around a circle of radius R (e.g., centered on equivalentpolar-angle increments), and some may be shifted from equal spacingaround the circle. In some embodiments, the nano-antennas may bearranged in a spiral configuration around a sample well. Additionally oralternatively, other configurations of nano-antennas are possible, suchas a matrix array around the sample well, a cross distribution, and stardistributions. Individual nano-antennas may be shapes other than acircle, such as square, rectangular, cross, triangle, bow-tie, annularring, pentagon, hexagon, polygons, etc. In some embodiments, thecircumference of an aperture or disc may be approximately an integermultiple of a fractional wavelength, e.g., (N/2)λ.

A nano-antenna array may direct emission energy from a sample intoconcentrated radiation lobes that have a spatial pattern dependent upona characteristic wavelength of the emission energy. When a sample emitsenergy, it may excite surface plasmons that propagate from the samplewell to the nano-antennas distributed around the sample well. Thesurface plasmons may then excite radiation modes or dipole emitters atthe nano-antennas that emit radiation perpendicular to the surface ofthe sample well layer. The phase of an excited mode or dipole at anano-antenna will depend upon the distance of the nano-antenna from thesample well. Selecting the distance between the sample well and anindividual nano-antenna controls the phase of radiation emitted from thenano-antenna. The spatial radiation mode excited at a nano-antenna willdepend upon the geometry and/or size of the nano-antenna. Selecting thesize and/or geometry of an individual nano-antenna controls the spatialradiation mode emitted from the nano-antenna. Contributions from allnano-antennas in the array and, in some instances the sample well, maydetermine an overall radiation lobe or lobes that form the radiationpattern. As may be appreciated, phase and spatial radiation mode emittedfrom an individual nano-antenna may depend upon wavelength, so that theoverall radiation lobe or lobes that form the radiation pattern willalso be dependent upon wavelength. Numerical simulations of theelectromagnetic fields may be employed to determine overall radiationlobe patterns for emission energies of different characteristicwavelengths.

The nano-antenna may comprise an array of holes or apertures in aconductive film. For example, the nano-antenna structure may be formedat the interface between a conductive sample well layer and anunderlying dielectric layer. The holes may comprise sets of holesdistributed in concentric circles surrounding a central point. In someembodiments, a sample well is located at the central point of the array,while in other embodiments the sample well may be off-center. Eachcircularly-distributed set of holes may comprise a collection ofdifferent diameters arranged smallest to largest around the circulardistribution. The hole diameters may be different between the sets(e.g., a smallest hole in one set may be larger than a smallest hole inanother set), and the location of the smallest hole may be oriented at adifferent polar angle for each set of circles. In some embodiments,there may be one to seven sets of the circularly-distributed holes in anano-antenna. In other embodiments, there may be more than seven sets.In some embodiments, the holes may not be circular, but may be anysuitable shape. For example, the holes may be ellipses, triangles,rectangles, etc. In other embodiments, the distribution of holes may notbe circular, but may create a spiral shape.

FIGS. 6-3A and 6-3B illustrate an exemplary nano-antenna structurecomprised of holes or apertures in a conductive layer. FIG. 6-3A shows atop planar view of the surface of an integrated device with a samplewell 6-108 surrounded by holes 6-122. The nano-antenna holes aredistributed approximately around a circle of radius R. In thisnon-limiting example, the hole diameters vary by incrementallyincreasing around the circumference of the circle of holes. FIG. 6-3Bshows a schematic elevation view of the nano-antenna shown in FIG. 6-3Aalong line B-B. A sample well layer 6-116 may comprise a conductor andinclude the sample well 6-108 and apertures 6-122 that are part of thenano-antenna structure. An adjacent layer 6-118 may be a dielectricmaterial and/or an optically transparent material.

In some embodiments, a nano-antenna structure may comprise a pluralityof disks. The disks of the nano-antenna structure may be formed asconductive disks protruding from a surface of a conductive material. Theconductive material may be adjacent an optically-transparent material,according to some embodiments. In some embodiments, the nano-antennasmay be distributed around a sample well. In some instances, thenano-antennas may be distributed around a sample well with their centersapproximately at a circle of radius R. A nano-antenna array may comprisemultiple sets of nano-antennas distributed approximately on additionalcircles of different radii around a sample well.

FIGS. 6-3C and 6-3D illustrate an exemplary embodiment of a nano-antennastructure comprising disks protruding from a conductive layer. FIG. 6-3Cshows a top planar view schematic of the surface of an integrated devicewith a sample well 6-208 surrounded by disks 6-224. The nano-antennadisks are distributed approximately around a circle of radius R. In thisnon-limiting example, two diameters are used for the disks and the disksalternate between these two diameters around the circumference of thecircle of nano-antenna. FIG. 6-3D shows a schematic elevation view ofthe nano-antenna shown in FIG. 6-3C along line D-D. A sample well layer6-216 may comprise a conductor and include the sample well 6-208 anddisks 6-224 that are part of the nano-antenna structure. The disks 6-224protrude from the sample well layer 6-216 by a certain distance. In someembodiments, the distance the disks extend from the sample well layermay vary within a nano-antenna structure. An adjacent layer 6-218 maycomprise a dielectric material and/or an optically-transparent material.The sample well layer 6-216 and the protruding disks may be a conductivematerial.

The holes and/or disks that form a nano-antenna structure may be anysuitable pattern or distribution such that emission energy from samplewell couples with one or more of the nano-antennas of the nano-antennastructure. Another example of a nano-antenna structure is illustrated inFIG. 6-4A, which represents a spiral pattern in which a nano-antenna maybe formed. A sample well may be located within a sample well layer atposition 6-308 with respect to nano-antenna structure 6-312. Surfaceplasmons may form in the area of the nano-antenna structure whenemission energy is emitted from the sample well. FIG. 6-4B illustratesresults from a numerical simulation of surface plasmons in the vicinityof a nano-antenna structure, according to some embodiments. The resultsalso show electromagnetic field intensity with the apertures of thenano-antenna. Other exemplary patterns and distributions ofnano-antennas that form a nano-antenna structure within a pixel areshown in FIG. 6-4C through FIG. 6-4E.

A nano-antenna structure may be used to distinguish emissions atdifferent characteristic wavelengths. The nano-antenna aperturestructure may produce radiation lobes that extend from the sample wellin different directions for emission energy of different characteristicwavelengths. The radiation lobes form a spatial distribution patternthat differs depending on the characteristic wavelength of the emissionenergy. Examples of possible spatial distribution patterns that form asa result of having a nano-antenna structure positioned underneath asample well is depicted in FIG. 6-5A through FIG. 6-5D. For example, alayer 6-906 within a pixel may contain a sample well with anano-aperture structure 6-902 positioned adjacent the sample well. Whenemission energy having a first characteristic wavelength is emitted by asample in the sample well, the emission energy couples with thenano-antennas in the nano-antenna structure which directs the emissionenergy into a first spatial distribution pattern 6-904 illustrated inFIG. 6-5A. Additionally, when emission energy having a secondcharacteristic wavelength is emitted by a sample in the sample well, asecond distribution pattern may form, such as the distribution pattern6-1004 shown in FIG. 6-5B. Similarly, FIG. 6-5C illustrates a thirdspatial distribution pattern 6-1104 for emission energy having a thirdcharacteristic wavelength, and FIG. 6-5D illustrates a fourth spatialdistribution pattern 6-1204 having a fourth characteristic wavelength.The different spatial distribution patterns may be detected by spatiallyseparated sensors within the pixel to differentiate among the first,second, third, and fourth characteristic wavelengths.

VI. B. Far Field Optics

Emission energy emitted from a sample in the sample well may betransmitted to the sensor of a pixel in a variety of ways, some examplesof which are described in detail below. Some embodiments may use opticaland/or plasmonic components to increase the likelihood that light of aparticular wavelength is directed to one or more segments of the sensor.The sensor may include multiple segments for simultaneously detectingemission energy of different wavelengths.

FIG. 6-6A is a schematic diagram of a single pixel of an integrateddevice according to some embodiments where at least one sorting elementis used to direct emission energy of a particular wavelength to arespective sensor segment, according to some embodiments. A sample well6-601 formed in a conductive material 6-603 receives a sample and mayemit emission energy 6-604. For clarity, details of any optical andplasmonic components at the sample well are not shown. The emissionenergy 6-604 travels through a dielectric material 6-605 until itreaches a sorting element 6-607. The sorting element 6-607 couples thewavelength of the emission energy 6-604 to a spatial degree of freedom,thereby separating the emission energy into its constituent wavelengthcomponents, referred to as sorted emission energy. FIG. 6-6B illustratesschematically the emission energy 6-604 being split into four sortedemission energy paths through a dielectric material 6-609, each of thefour paths associated with a sub-sensor 6-611 through 6-614 of thepixel. In this way, each sensor segment may be associated with adifferent portion of the spectrum, forming a spectrometer for each pixelof the integrated device.

Any suitable sorting element 6-607may be used to separate the differentwavelengths of the emission energy. Embodiments may use optical orplasmonic elements. Examples of optical sorting elements include, butare not limited to, holographic gratings, phase mask gratings, amplitudemask gratings, frequency selective surfaces, diffractive opticalelements, and offset Fresnel lenses. Examples of plasmonic sortingelements include, but are not limited to phased nano-antenna arrays, andplasmonic quasi-crystals.

FIG. 6-6B is a schematic diagram of a single pixel of an integrateddevice according to some embodiments where at least one filteringelement is used to direct emission energy of a particular wavelength toa respective sub-sensor and prevent emission energy of other wavelengthsfrom reaching the sub-sensor. Where the components of FIG. 6-6B aresimilar to those of FIG. 6-6A, the same reference numerals are used. Asample well 6-601 formed in a conductive material 6-603 receives asample and may emit emission energy 6-604. For clarity, details ofoptical and plasmonic components at the sample well are not shown. Theemission energy 6-604 travels through a dielectric material 6-605 untilit reaches one of the filtering elements 6-621 through 6-624. Thefiltering elements 6-621 through 6-624, may each be associated with aparticular segment of a sensor 6-611 through 6-614, and are eachconfigured to transmit emission energy of a respective wavelength andreject emission energy of other wavelengths by absorbing the emissionenergy (not illustrated in FIG. 6-6B) and/or reflecting the emissionenergy. After passing through a respective filtering element, thefiltered emission energy travels through a dielectric material 6-609 andimpinges on a corresponding sub-sensor 6-611 through 6-614 of the pixel.In this way, each sub-sensor is associated with a different portion ofthe spectrum, forming a spectrometer for each pixel of the integrateddevice.

Any suitable filtering elements may be used to separate the differentwavelengths of the emission energy. Embodiments may use optical orplasmonic filtering elements. Examples of optical sorting elementsinclude, but are not limited to, reflective multilayer dielectricfilters or absorptive filters. Examples of plasmonic sorting elementsinclude, but are not limited to frequency selective surfacesconfigurationed to transmit energy at a particular wavelength andphotonic band-gap crystals.

Alternatively, or in addition to the above mentioned sorting elementsand filtering elements, additional filtering elements may be placedadjacent to each sub-sensor 6-61 through 6-614. The additional filteringelements may include a thin lossy film configured to create constructiveinterference for emission energy of a particular wavelength at thesensor or sensor segment for a particular wavelength. The thin lossyfilm may be a single or multi-layer film. The thin lossy film may bemade from any suitable material. For example, the thin lossy film may bemade from a material where the index of refraction n is approximatelythe same order of magnitude as the extinction coefficient k. In otherembodiments, the thin lossy film may be made from a material where theindex of refraction n is within about two orders of magnitude differencefrom the value of the extinction coefficient k of the material.Non-limiting examples of such materials at visible wavelengths aregermanium and silicon.

The thin lossy film may be any suitable thickness. In some embodiments,the thin lossy film may be 1-45 nm thick. In other embodiments, the thinlossy film may be 15-45 nm thick. In still other embodiments, the thinlossy film may be 1-20 nm thick. FIG. 6-7A illustrates an embodimentwhere the thin lossy films 6-711 through 6-714 each have a differentthickness determined at least in part by the wavelength that isassociated with each sub-sensor 6-61 through 6-614. The thickness of thefilm determines, at least in part, a distinct wavelength that willselectively pass through the thin lossy film to the sub-sensor. Asillustrated in FIG. 6-7A, thin lossy film 6-711 has a thickness d1, thinlossy film 6-712 has a thickness d2, thin lossy film 6-713 has athickness d3, and thin lossy film 6-714 has a thickness d4. Thethickness of each subsequent thin lossy film is less than the previousthin lossy film such that d1>d2>d3>d4.

Additionally, or alternatively, the thin lossy films may be formed of adifferent material with a different properties such that emission energyof different wavelengths constructively interfere at each respectivesub-sensor. For example, the index of refraction n and/or the extinctioncoefficient k may be selected to optimize transmission of emissionenergy of a particular wavelength. FIG. 6-7B illustrates thin lossyfilms 6-721 through 6-724 with the same thickness but each thin lossyfilm is formed from a different material. In some embodiments, both thematerial of the thin lossy films and the thickness of the thin lossyfilms may be selected such that emission energy of a desired wavelengthconstructively interferes and is transmitted through the film.

Any one or more of the foregoing embodiments of emission-couplingelements may be included in an embodiment of an integrated device.

VII. Sensors

Various embodiments of sensors, sensor operation, and signal processingmethods have been contemplated by the inventors. According to someembodiments, a sensor 3-260 at a pixel may comprise any suitable sensorcapable of receiving emission energy from one or more tags in the samplewell, and producing one or more (e.g., at least 2, 3, or 4) electricalsignals representative of the received emissions. In some embodiments, asensor may comprise at least one, two, three, or four photodetectors.Each photodetector may comprise a p-n junction formed in a semiconductorsubstrate. FIG. 7-1A depicts just one embodiment of a sensor that may befabricated within a pixel 3-100 of an integrated device.

According to some embodiments, a sensor 3-260 may be formed at eachactive pixel 3-100 of an integrated device. The sensor may be centeredabout a sample well 3-210, and spaced from the sample well by a distancebetween approximately 1 micron and approximately 20 microns. There maybe one or more transparent layers 7-110 between the sample well and thesensor, so that emission from the sample well may travel to the sensorwithout significant attenuation. The sensor 3-260 may be formed in asemiconductor substrate 7-120 at a base of the pixel, according to someembodiments, and be located on a same side of the sample well as theexcitation source (not shown).

The sensor may comprise one or more semiconductor junction photodetectorsegments. Each semiconductor junction may comprise a well of a firstconductivity type. For example, each semiconductor junction may comprisean n-type well formed in a p-type substrate, as depicted in the drawing.According to some embodiments, a sensor 3-260 may be arranged as abulls-eye detector 7-162, as depicted in the plan view of FIG. 7-1B. Afirst photodetector 7-124 may be located at a center of the sensor, anda second annular photodetector 7-122 may surround the centerphotodetector. Electrical contacts to the wells may be made throughconductive traces 7-134 formed at a first or subsequent metallizationlevel and through conductive vias 7-132. There may be a region of highlydoped semiconductor material 7-126 at contact regions of the vias. Insome embodiments, a field oxide 7-115 may be formed at surfaces betweenthe photodetectors and or may cover a portion of each photodetector. Insome implementations, there may be additional semiconductor devices7-125 (e.g., transistors, amplifiers, etc.) formed within the pixeladjacent to the sensor 3-260. There may be additional metallizationlevels 7-138, 7-136 within the pixel.

In some implementations, a metallization levels 7-136 may extend acrossa majority of the pixel and have an opening below the sample well 3-210,so that emission from the sample well can reach the sensor. In somecases, a metallization level 7-136 may serve as a reference potential ora ground plane, and additionally serve as an optical block to prevent atleast some background radiation (e.g., radiation from an excitationsource or from the ambient environment) from reaching the sensor 3-260.

As depicted in FIG. 7-1A and FIG. 7-1B, a sensor 3-260 may be subdividedinto a plurality of photodetector segments 7-122, 7-124 that arespatially and electrically separated from each other. In someembodiments, segments of a sensor 3-260 may comprise regions ofoppositely-doped semiconductor material. For example, a first chargeaccumulation well 7-124 for a first sensor segment may be formed bydoping a first region of a substrate to have a first conductivity type(e.g., n-type) within the first well. The substrate may be p-type. Asecond charge accumulation well 7-122 for a second sensor segment may beformed by doping a second region of the substrate to have the firstconductivity type within the second well. The first and second wells maybe separated by a p-type region of the substrate.

The plurality of segments of the sensor 3-260 may be arranged in anysuitable way other than a bulls-eye layout, and there may be more thantwo segments in a sensor. For example, in some embodiments, a pluralityof photodetector segments 7-142 may be laterally separated from oneanother to form a stripe sensor 7-164, as depicted in FIG. 7-1C. In someembodiments, a quadrant sensor 7-166 may be formed by arranging thesegments 7-144 in a quad pattern, as depicted in FIG. 7-1D. In someimplementations, arc segments 7-146 may be formed in combination with abulls-eye pattern, as depicted in FIG. 7-1E, to form an arc-segmentedsensor 7-168. Another sensor configuration may comprise pie-piecesections, which may include individual sensors arranged in separatesections of a circle. In some cases, sensor segments may be arrangedsymmetrically around a sample well 3-210 or asymmetrically around asample well. The arrangement of sensor segments is not limited to onlythe foregoing arrangements, and any suitable distribution of sensorsegments may be used.

The inventors have found that a quadrant sensor 7-166, pie-sectorsensor, or similar sector sensor can scale to smaller pixel sizes morefavorably than other sensor configurations. Quadrant and sectordetectors may consume less pixel area for a number of wavelengthsdetected and active sensor area. Quadrant and sector detectors may beused in combination with nano-antenna arrays or surface-plasmonstructures to produce distinct spatial distribution patterns that arediscernable by the detectors. Sensors may be arranged in variousgeometric configurations. In some examples, sensors are arranged in asquare configuration or hexagonal configuration.

Sensors of the present disclosure may be independently (or individually)addressable. An individually addressable sensor is capable of detectingemission from a corresponding sample well and providing output signalsindependent of other sensors. An individually addressable sensor may beindividually readable.

In some embodiments, a stacked sensor 7-169 may be formed by fabricatinga plurality of separated sensor segments 7-148 in a vertical stack, asdepicted in FIG. 7-1F. For example, the segments may be located oneabove the other, and there may, or may not, be insulating layers betweenthe stacked segments. Each vertical layer may be configured to absorbemission energy of a particular energy, and pass emission at differentenergies. For example, a first detector may absorb and detectshorter-wavelength radiation (e.g., blue-wavelength radiation belowabout 500 nm from a sample). The first detector may pass green- andred-wavelength emissions from a sample. A second detector may absorb anddetect green-wavelength radiation (e.g., between about 500 nm and about600 nm) and pass red emissions. A third detector may absorb and detectthe red emissions. Reflective films 7-149 may be incorporated in thestack, in some embodiments, to reflect light of a selected wavelengthband back through a segment. For example, a film may reflectgreen-wavelength radiation that has not been absorbed by the secondsegment back through the second segment to increase its detectionefficiency.

In some embodiments with vertically-stacked sensor segments,emission-coupling components may not be included at the sample well toproduce distinct spatial distribution patterns of sample emission thatare dependent on emission wavelength. Discernment of spectrallydifferent emissions may be achieved with a vertically-stacked sensor7-169 by analyzing the ratio of signals from its stacked segment,according to some embodiments.

In some embodiments, segments of a sensor 3-260 are formed from silicon,though any suitable semiconductor (e.g., Ge, GaAs, SiGe, InP, etc.) maybe used. In some embodiments, a sensor segment may comprise an organicphotoconductive film. In other embodiments, quantum dot photodetectorsmay be used for sensor segments. Quantum dot photodetectors may respondto different emission energies based on the size of the quantum dot. Insome embodiments, a plurality of quantum dots of varying sizes may beused to discriminate between different emission energies or wavelengthsreceived from the sample well. For example, a first segment may beformed from quantum dots having a first size, and a second segment maybe formed from quantum dots having a second size. In variousembodiments, sensors 2-260 may be formed using conventional CMOSprocesses.

As described above, emission-coupling components may be fabricatedadjacent the sample well in some embodiments. The emission-couplingcomponents can alter emission from a sample within the sample well 3-210to produce distinct spatial distribution patterns of sample emissionthat are dependent on emission wavelength. FIG. 7-2A depicts an exampleof a first spatial distribution pattern 7-250 that may be produced froma first sample at a first wavelength. The first spatial distributionpattern 7-250 may have a prominent central lobe directed toward acentral segment of a bulls-eye sensor 7-162, for example. As just oneexample, such a pattern 7-250 may be produced from a sample wellsurrounded by a circular grating 7-220 emission-coupling structure,where the sample emits at a wavelength of about 663 nm. A projectedpattern 7-252 incident on the sensor may appear as illustrated in FIG.7-2B.

FIG. 7-2C depicts a spatial distribution pattern 7-260 that may beproduced from a second sample emitting at a second wavelength from thesame sample well, according to some embodiments. The second spatialdistribution pattern 7-260 may comprise two lobes of radiation anddiffer from the first spatial distribution pattern 7-250. A projectedpattern 7-262 of the second spatial distribution pattern 7-260 mayappear as depicted in FIG. 7-2D, according to some embodiments. As justone example, a second spatial distribution pattern 7-260 may be producedfrom the same sample well surrounded by the circular grating 7-220emission-coupling structure, where the sample emits at a wavelength ofabout 687 nm.

The segments of a sensor 3-260 may be arranged to detect particularemission energies, according to some embodiments. For example,emission-coupling structures adjacent the sample well and segments of asensor may be configurationed in combination to increase signaldifferentiation between particular emission energies. The emissionenergies may correspond to selected tags that will be used with theintegrated device. As an example, a bulls-eye sensor 7-162 could haveits segments sized and/or located to better match the projected patterns7-260, 7-262 from a sample, so that regions of higher intensity fallmore centrally within active segments of the sensor. Alternatively oradditionally, emission-coupling structures may be configurationed toalter the projected patterns 7-260, 7-262 so that intense regions fallmore centrally within segments of the sensor.

Although a sensor 3-260 may comprise two segments, it is possible insome embodiments to discern more than two spectrally-distinct emissionbands from a sample. For example, each emission band may produce adistinct projected pattern on the sensor segments and yield a distinctcombination of signals from the sensor segments. The combination ofsignals may be analyzed to discern and identify the emission band. FIG.7-2E through FIG. 7-2H represent results from numerical simulations ofsignal sets from a two-segment sensor 3-260 exposed to four distinctemission patterns from four different emitters. The emission patternswere simulated as being produced at four wavelengths (565 nm, 595 nm,663 nm, 687 nm) from a sample well having a circular grating formedadjacent the sample well. As can be seen, each combination of signals(or signal set) from the two sensor segments is distinct, and can beused to discriminate between emitters at the four wavelengths. For thesimulation, because the outer detector segment of the bulls-eye sensor7-162 had a larger area, more signal was integrated for that detector.Additionally, light that impinged on an area between the detectorsgenerated carriers that may drift towards either detector segment andcontribute to signals from both segments.

In some embodiments, there may be N photodetector segments per pixel,where N may be any integer value. In some embodiments, N may be greaterthan or equal to 1 and less than or equal to 10. In other embodiments, Nmay be greater than or equal to 2 and less than or equal to 5. Thenumber M of discernible sample emissions (e.g., distinct emissionwavelengths from different luminescent tags) that may be detected by theN detectors may be equal to or greater than N. The discernment of Msample emissions may be achieved by evaluating the ratio of signals fromeach sensor segment, according to some embodiments. In someimplementations, the ratio, sum and/or amplitudes of the receivedsignals may be measured and analyzed to determine a characteristicwavelength of emission from the sample well.

In some embodiments, more than one emitter may emit at differentcharacteristic wavelengths in a given time window within a sample well3-210. A sensor 3-260 may simultaneously detect signals from multipleemissions at different wavelengths and provide the summed signal fordata processing. In some implementations, multi-wavelength emission maybe distinguishable as another set of signal values from the sensorsegments (e.g., signal values different from those shown in FIG. 7-2Ethrough FIG. 7-2H). The signal values may be analyzed to discern thatmulti-wavelength emission has occurred and to identify a particularcombination of emitters associated with the emissions.

The inventors have also contemplated and analyzed a bulls-eye sensorhaving at least two, three, or four concentric segments. Signal setsfrom the segments are plotted in FIG. 7-2I and FIG. 7-2J for the sameemission conditions associated with FIG. 7-2G and FIG. 7-2H,respectively. The four-segment bulls-eye sensor also shows discernablesignals that may be analyzed to identify a particular emitter within thesample well.

When wavelength filtering is used at each sensor segment, or thespectral separation is high, each segment of a sensor may detectsubstantially only a selected emission band. For example, a firstwavelength may be detected by a first segment, a second wavelength maybe detected by a second segment, and a third wavelength may be detectedby a third segment.

Referring again to FIG. 7-1A, there may be additional electroniccircuitry 7-125 within a pixel 2-205 that may be used to collect andreadout signals from each segment of a sensor 3-260. FIG. 7-3A and FIG.7-3D depict circuitry that may be used in combination with amulti-segment sensor, according to some embodiments. As an example,signal collection circuitry 7-310 may comprise three transistors foreach sensor segment. An arrangement of the three transistors is depictedin FIG. 7-3B, according to some implementations. A signal level at acharge accumulation node 7-311 associated with each segment may be resetby a reset transistor RST prior to a charge-accumulation period, and asignal level for the segment (determined by the amount of charge at thecharge accumulation node) may be read out with a read transistor RDduring and/or at the conclusion of a charge-accumulation period. Signalsmay be provided to a processor (not shown) for analysis to discern thedetection of M different emission wavelengths from the sample detectedby N spatially-separated detectors, as described above.

The pixel circuitry may further include amplification and correlateddouble-sampling circuitry 7-320, according to some embodiments. Theamplification and double-sampling circuitry may comprise transistorsconfigured to amplify signals from the sensor segments as well astransistors configured to reset the voltage level at thecharge-accumulation node and to read a background, or “reset”, signal atthe node when no emission radiation is present on the sensor (e.g.,prior to application of excitation energy at the sample well) and toread a subsequent emission signal, for example.

According to some embodiments, correlated double sampling is employed toreduce background noise by subtracting a background or reset signallevel from the detected emission signal level. The collected emissionsignal and background signal associated with each segment of the sensormay be read out onto column lines 7-330. In some embodiments, anemission signal level and background signal are time-multiplexed onto acommon column line. There may be a separate column line for each sensorsegment. Signals from the column lines may be buffered and/or amplifiedwith amplification circuitry 7-340 (which may be located outside of anactive pixel array), and provided for further processing and analysis.In some embodiments the subtraction of the double-sampled signals iscalculated off-chip, e.g., by a system processor. In other embodiments,the subtraction may be performed on chip or in circuitry of the baseinstrument.

Some embodiments of correlated double sampling may operate by selectinga row to sample, wherein the sensors associated with the row haveintegrated signal charges over a sampling period and contain signallevels. The signal levels may be simultaneously read out onto thecolumns lines. After sampling the integrated signal levels, all thepixels in the selected row may be reset and immediately sampled. Thisreset level may be correlated to the next integrated signal that startsaccumulating after the reset is released, and finishes integrating aframe time later when the same row is selected again. In someembodiments, the reset values of the frame may be stored off-chip sothat when the signals have finished integrating and have been sampled,the stored correlated reset values can be subtracted.

In some embodiments, a sensor 3-260 with more than two segments mayrequire additional circuitry. FIG. 7-3C depicts signal-collection 7-312,amplification 7-320, and double-sampling circuitry associated with aquad sensor. According to some embodiments, signals from two or moresegments may be time-multiplexed onto a common signal channel at thepixel, as depicted in the drawing. The time-multiplexed signals mayinclude sampled background signals for each segment for noisecancellation. Additionally, the signals from two or more segments may betime-multiplexed onto a common column line.

According to some embodiments, temporal signal-acquisition techniquesmay be used to reduce background signal levels from an excitation sourceor sources, and/or discern different emissions from different emittersassociated with a sample. FIG. 7-4A depicts fluorescent emission anddecay from two different emitters that may be used to tag a sample,according to some embodiments. The two emissions have appreciablydifferent time-decay characteristics. A first time-decay curve 7-410from a first emitter may correspond to a common fluorescent moleculesuch as rhodamine. A second time-decay curve 7-420 may be characteristicof a second emitter, such as a quantum dot or a phosphorescent emitter.Both emitters exhibit an emission-decay tail that extends for some timeafter initial excitation of the emitter. In some embodiments,signal-collection techniques applied during the emission-decay tail maybe timed to reduce a background signal from an excitation source, insome embodiments, and to distinguish between the emitters, in someembodiments.

According to some implementations, time-delayed sampling may be employedduring the emission-decay tail to reduce a background signal due toradiation from an excitation source. FIG. 7-4B and FIG. 7-4C illustratetime-delay sampling, according to some embodiments. FIG. 7-4B depictsthe temporal evolution of an excitation pulse 7-440 of excitationradiation from an excitation source, and a subsequent emission pulse7-450 that may follow from a sample that is excited within the samplewell. The excitation pulse 7-440 may result from driving the excitationsource with a drive signal 7-442 for a brief period of time, as depictedin FIG. 7-4C. For example, the drive signal may begin at a first time t₁and end at a second time t₂. The duration of the drive signal (t₂-t₁)may be between about 1 picosecond and about 50 nanoseconds, according tosome embodiments, though shorter durations may be used in someimplementations.

At a time t₃ following termination of the drive signal for theexcitation source, a sensor 3-260 (or sensor segment) at the pixel maybe gated to accumulate charge at a charge accumulation node 7-311 (withreference to FIG. 7-3B) during a second time interval extending from atime t₃ to a time t₄. The second time interval may be between about 1nanosecond and about 50 microseconds, according to some embodiments,though other durations may be used in some implementations. As can beseen in reference to FIG. 7-4B, a charge accumulation node will collectmore signal charges due to the emitting sample then due to theexcitation source. Accordingly, an improved signal-to-noise ratio may beobtained.

Referring again to FIG. 7-4A, because of the different temporal emissioncharacteristics of the emitters, corresponding signals at a sensor maypeak at different times. In some implementations, signal-acquisitiontechniques applied during the emission-decay tail may be used to discerndifferent emitters. In some embodiments, temporal detection techniquesmay be used in combination with spatial and spectral techniques (asdescribed above in connection with FIG. 7-2, for example) to discerndifferent emitters.

FIG. 7-4D through FIG. 7-4H illustrate how double-sampling at a sensor,or sensor segment, can be used to distinguish between two emittershaving different temporal emission characteristics. FIG. 7-4D depictsemission curves 7-470, 7-475 associated with a first emitter and secondemitter, respectively. As an example, the first emitter may be a commonfluorophore such as rhodamine, and the second emitter may be a quantumdot or phosphorescent emitter.

FIG. 7-4E represents dynamic voltage levels at a charge accumulationnode 7-311 that may occur in response to the two different emissioncharacteristics of FIG. 7-4D. In the example, a first voltage curve7-472 corresponding to the fluorescent emitter may change more rapidly,because of the shorter emission span, and reach its maximum (or minimum,depending on the polarity of the node) at a first time t₁. The secondvoltage curve 7-477 may change more slowly due to the longer emissioncharacteristics of the second emitter, and reach its maximum (orminimum) at a second time t₂.

In some embodiments, sampling of the charge-accumulation node may bedone at two times t₃, t₄ after the sample excitation, as depicted inFIG. 7-4F. For example, a first read signal 7-481 may be applied to readout a first voltage value from the charge-accumulation node at a firsttime t₃. Subsequently, a second read signal 7-482 may be applied to readout a second voltage value from the charge-accumulation node at a secondtime t₄ without resetting the charge-accumulation node between the firstread and second read. The first read and second read at times t₃ and t₄may occur during a same charge-accumulation period for the sensor duringthe emission from the sample well. An analysis of the two sampled signalvalues may then be used to identify which of the two emitters providedthe detected signal levels.

FIG. 7-4G depicts an example of a first signal set from the first readand second read that may be obtained for the first emitter having anemission curve 7-470 as depicted in FIG. 7-4D. FIG. 7-4H depicts anexample of a second signal set from the first read and second read thatmay be obtained for the second emitter having an emission curve 7-475 asdepicted in FIG. 7-4D. For example the sampling sequence shown in FIG.7-4F for the first emitter will sample the curve 7-472 and obtainapproximately the same values at the two read times. In the case of thesecond emitter, the sampling sequence depicted in FIG. 7-4F samples twodifferent values of the curve 7-477 at the two read times. The resultingpairs of signals from the two read times distinguish between the twoemitters, and can be analyzed to identify each emitter. According tosome embodiments, double sampling for background subtraction may also beexecuted to subtract a background signal from the first and second readsignals.

In operation, sensors 2-260 of an integrated device may be subjected toa wavelength calibration procedure prior to data collection from aspecimen to be analyzed. The wavelength calibration procedure mayinclude subjecting the sensors to different known energies havingcharacteristic wavelengths that may, or may not, correspond tofluorophore wavelengths that may be used with an integrated device. Thedifferent energies may be applied in a sequence so calibration signalscan be recorded from the sensors for each energy. The calibrationsignals may then be stored as reference signals, that may be used toprocess real data acquisition and to determine what emission wavelengthor wavelengths are detected by the sensors.

According to some embodiments, a sensor may comprise a semiconductorjunction formed adjacent the sample well 3-210. The semiconductorjunction may be as depicted in FIG. 4-5B or FIG. 4-5D, for example. Insome implementations, the semiconductor junction may be formed as amultilayer structure, and the sample well may be formed in themultilayer structure, as depicted in FIG. 3-7F, for example. In someembodiments, an excited sample may non-radiatively transfer emissionenergy to a semiconductor junction formed adjacent the sample well viaFRET or DET, creating excitons at the semiconductor junction. Thesemiconductor junction may comprise a p-n or p-i-n junction thatconverts the received energy to an electrical signal that is detected byCMOS circuitry associated with the sample well. In some implementations,a quantum dot or molecule may be attached to the semiconductor junctionvia a linker and may participate in non-radiative energy transfer froman excited sample to the semiconductor junction.

Any one or more of the foregoing embodiments of sensors may be includedin an embodiment of an integrated device.

VIII. Instrument Operation

The instrument 2-120 may be controlled using software and/or hardware.For example, the instrument may be controlled using a processing device1-123, such as an ASIC, an FPGA and/or a general purpose processorexecuting software.

FIG. 8-1 illustrates a flowchart of operation of the instrument 2-120according to some embodiments. After a user has acquired a specimen toanalyze, the user begins a new analysis at act 8-101. This may be doneby providing an indication to the instrument 2-120 via the userinterface 2-125 by, e.g., pressing a button. At act 8-103, theinstrument 2-120 checks whether the integrated device 2-110 from apreviously performed analysis is still inserted in the instrument 2-120.If it is determined that an old integrated device is present, then thepower to excitation source may be turned off at act 8-105, the user isprompted at act 8-107 to eject the previous integrated device using anindicator of the user interface 2-125 and the instrument 2-120 waits forthe old integrated device to be ejected at act 8-109.

When the previous integrated device is ejected by the user, or if theinstrument 2-120 determined at act 8-103 that the previous integrateddevice was already removed, the user is prompted to insert a newintegrated device 2-110 for the new analysis at act 8-111. Theinstrument 2-120 then waits for the new integrated device 2-110 to beinserted at act 8-113. When the user inserts the new integrated device,the user is prompted at act 8-115 by an indicator of the user interface2-125 to place the specimen to be analyzed onto the exposed top surfaceof the integrated device 2-110 and also prompted to close the lid on theinstrument 2-120. The instrument 2-120 then waits for the lid to beclosed at act 8-117. When the lid is closed by the user, at act 8-119the excitation source may be driven to produce excitation energy forexciting the sample portions of the specimen present in the sample wellsof the integrated device 2-110. At act 8-121, the emission energy fromthe samples is detected by the sensor 2-122 and data from the sensor2-122 is streamed to the processing device 2-123 for analysis. In someembodiments, the data may be streamed to external computing device2-130. At act 2-123, the instrument 2-120 checks whether the dataacquisition is complete. The data acquisition may be complete after aparticular length of time, a particular number of excitation pulses fromthe excitation source or one a particular target has been identified.When the data acquisition is completed, the data analysis is finished at8-125.

FIG. 8-2 illustrates an example self-calibration routine according tosome embodiments. The calibration routine may be executed at anysuitable time prior to the analysis of a specimen. For example, it maybe done once by the manufacturer for each instrument prior to shipmentto the end user. Alternatively, the end user may perform a calibrationat any suitable time. As discussed above, the instrument 2-120 iscapable of distinguishing between emission energy having differentwavelengths emitted from different samples. The instrument 2-120 and/orcomputing device 2-130 may be calibrated with calibration associatedwith each particular color of light associated with, for example, aluminescent tag used to tag molecules of a specimen being analyzed. Inthis way, the precise output signal associated with a particular colormay be determined.

To calibrate the device, a calibration specimen associated with a singleluminescent tag is provided to the instrument 2-120 one at a time. Theself-calibration begins at act 8-201 when a user places a specimencomprising luminescent tags that emit emission energy of a singlewavelength on an integrated device 2-110 and inserts the integrateddevice 2-110 into the instrument 2-120. Using the user interface 2-125,the user instructs the instrument 2-120 to begin the self-calibration.In response, at act 8-203, the instrument 2-120 runs the calibrationanalysis by illuminating the assay chop 2-110 with excitation energy andmeasuring the single wavelength emission energy from the calibrationspecimen. The instrument 2-120 may then, at act 8-205, save thedetection pattern measured on the array of sub-sensors of the sensor2-122 for each pixel of the sensor array. The detection pattern for eachluminescent tag may be considered a detection signature associated withthe luminescent tag. In this way, the signatures may be used as atraining data set used to analyze the data received from unknown samplesanalyzed in subsequent analysis runs.

The above calibration routine may then be executed for every calibrationspecimen associated with a single luminescent tag. In this way, eachsensor 2-122 of the array of pixels is associated with calibration datathat may be used to determine the luminescent tag present in a samplewell during a subsequent analysis implemented at act 8-207 after thecompetition of the calibration routine.

FIG. 8-3 further illustrates how the calibration data may be acquiredand used to analyze the data according to some embodiments. At act 8-301calibration data is obtained from the sensors. This may be done usingthe aforementioned self-calibration routine. At act 8-303, atransformation matrix is generated based on the calibration data. Thetransformation matrix maps sensor data to the emission wavelength of asample and is a m x n matrix, where m is the number of luminescent tagswith different emission wavelengths and n is the number of sub-sensorsused to detect the emission energy per pixel. Thus, each column of thetransformation matrix represents the calibration values for the sensor.For example, if there are four sub-sensors per pixel and five differentluminescent tags, then the transformation matrix is a 4×5 matrix (i.e.,four rows and five columns) and each column is associated with adifferent luminescent tag, the values in the column corresponding to themeasured values obtained from the sub-sensors during theself-calibration routine. In some embodiments, each pixel may have itsown transformation matrix. In other embodiments, the calibration datafrom at least some of the pixels may be averaged and all the pixels maythen use the same transformation matrix based on the averaged data.

At act 8-305, the analysis data associated with a bioassay is obtainedfrom the sensors. This may be done in any of the ways described above.At act 8-307, the wavelength of the emission energy and/or the identityof the luminescent tag may be determined using the transformation matrixand the analysis data. This may be done in any suitable way. In someembodiments, the analysis data is multiplied by the pseudo-inverse ofthe transformation matrix, resulting in a m×1 vector. The luminescenttag associated with the vector component with the maximum value may thenbe identified as the luminescent tag present in the sample well.Embodiments are not limited to this technique. In some embodiments, toprevent possible pathologies that may arise when the inverse of a matrixwith small values is taken, a constrained optimization routine, such asa least square method or a maximum likelihood technique, may beperformed to determine the luminescent tag present in the sample well.

The foregoing method of using the calibration data to analyze data fromthe sensors may be implement by any suitable processor. For example,processing device 2-123 of the instrument 2-120 may perform theanalysis, or computing device 2-130 may perform the analysis.

FIG. 8-2 illustrates the base instrument control of the aforementionedcorrelated double sampling of the pixels of the integrated bioanalysisdevice 212 according to some embodiments. At the start of a new frame ofdata acquisition, a row shift register is reset. The pixel reset valuefrom the previous frame is read by incrementing the column register.Simultaneously the current frames pixel sample levels are stored withinthe reading element on the integrated device. Once the desired number ofcolumns to be measured is reached, the column register is reset. Thenthe pixel sample levels from the current frame are read by incrementingthe column register and outputting the sample values eight pixels at atime to a buffer, in some embodiments the first frame of sample levelscan be discarded. The buffer can be located off integrated device inmemory or in some embodiments it can be stored locally on the integrateddevice. Once the number of columns to be measured is met the rowregister is incremented. This processes is repeated until a frame iscompleted. Upon finishing a frame of data the processes is started againwith the change that the frames sample levels are subtracted from theprevious frames reset levels.

IX. Conclusion

Having thus described several aspects of several embodiments of anintegrated bioanalysis device, it is to be appreciated that variousalterations, modifications, and improvements will readily occur to thoseskilled in the art. Such alterations, modifications, and improvementsare intended to be part of this disclosure, and are intended to bewithin the spirit and scope of the invention. While the presentteachings have been described in conjunction with various embodimentsand examples, it is not intended that the present teachings be limitedto such embodiments or examples. On the contrary, the present teachingsencompass various alternatives, modifications, and equivalents, as willbe appreciated by those of skill in the art.

For example, embodiments may be modified to include any configuration ofexcitation source, energy-coupling components, target volume, andenergy-collection components described above. Moreover, the integrateddevice may be used to quantitatively analyze non-biological samples.Additionally, various optical elements described herein, such aswaveguides, reflectors, and cavities, may be replaced with theirphotonic crystal equivalent; any metal material may be replaced with ahighly degenerately doped semiconductor; graphene may be used in placeof metals and/or semiconductors; phosphorescence may be used instead ofluminescence; and any single functional layer may be replaced with aplurality of functional layers.

While various inventive embodiments have been described and illustrated,those of ordinary skill in the art will readily envision a variety ofother means and/or structures for performing the function and/orobtaining the results and/or one or more of the advantages described,and each of such variations and/or modifications is deemed to be withinthe scope of the inventive embodiments described. More generally, thoseskilled in the art will readily appreciate that all parameters,dimensions, materials, and configurations described are meant to beexamples and that the actual parameters, dimensions, materials, and/orconfigurations will depend upon the specific application or applicationsfor which the inventive teachings is/are used. Those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific inventive embodimentsdescribed. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, inventiveembodiments may be practiced otherwise than as specifically describedand claimed. Inventive embodiments of the present disclosure may bedirected to each individual feature, system, system upgrade, and/ormethod described. In addition, any combination of two or more suchfeatures, systems, and/or methods, if such features, systems, systemupgrade, and/or methods are not mutually inconsistent, is includedwithin the inventive scope of the present disclosure.

Further, though some advantages of the present invention may beindicated, it should be appreciated that not every embodiment of theinvention will include every described advantage. Some embodiments maynot implement any features described as advantageous. Accordingly, theforegoing description and drawings are by way of example only.

All literature and similar material cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, and web pages, regardless of the format of suchliterature and similar materials, are expressly incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The section headings used are for organizational purposes only and arenot to be construed as limiting the subject matter described in any way.

The terms “program” or “software” may be used in the present disclosureto refer to computer code or set of computer-executable instructionsthat can be employed to program a computer or other processor toimplement various aspects of the present technology as discussed above.Additionally, it should be appreciated that according to one aspect ofthis embodiment, one or more computer programs that when executedperform methods of the present technology need not reside on a singlecomputer or processor, but may be distributed in a modular fashionamongst a number of different computers or processors to implementvarious aspects of the present technology.

The term “associated with,” when used in connection with datastructures, may be used to describe a combination of data structures insome embodiments. For example, first data associated with second datamay mean adding the first data to a data record containing the seconddata, or vice versa. “Associated with” may mean establishing arelational data structure between first and second data in someembodiments. For example, first data may be entered in a table oraugmented with an identifier that cross-references or links the firstdata to second data, even though the first and second data may be storedin different data stores.

The term “transmit,” when used in connection with data structures, maybe used to describe one or more acts of retrieving data, preparing thedata in a format suitable for transmission, identifying at least onedestination for the data, and providing the data to a data-transmissiondevice.

Where user-interactive displays are described, active text or buttonsmay alter their appearance when selected or clicked on by a user. Forexample, active text or buttons may change color or be highlighted inany suitable manner when selected, so as to indicate that the text orbutton has been selected.

Also, the technology described may be embodied as a method, of which atleast one example has been provided. The acts performed as part of themethod may be ordered in any suitable way. Accordingly, embodiments maybe constructed in which acts are performed in an order different thanillustrated, which may include performing some acts simultaneously, eventhough shown as sequential acts in illustrative embodiments.

All definitions, as defined and used, should be understood to controlover dictionary definitions, definitions in documents incorporated byreference, and/or ordinary meanings of the defined terms.

The indefinite articles “a” and “an,” as used in the specification andin the claims, unless clearly indicated to the contrary, should beunderstood to mean “at least one.”

The phrase “and/or,” as used in the specification and in the claims,should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used shall only be interpreted as indicating exclusive alternatives(i.e. “one or the other but not both”) when preceded by terms ofexclusivity, such as “either,” “one of,” “only one of,” or “exactly oneof.” “Consisting essentially of,” when used in the claims, shall haveits ordinary meaning as used in the field of patent law.

As used in the specification and in the claims, the phrase “at leastone,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively.

The claims should not be read as limited to the described order orelements unless stated to that effect. It should be understood thatvarious changes in form and detail may be made by one of ordinary skillin the art without departing from the spirit and scope of the appendedclaims. All embodiments that come within the spirit and scope of thefollowing claims and equivalents thereto are claimed.

1. An integrated device for analyzing a plurality of samples inparallel, the device comprising: a plurality of pixels formed on asubstrate; a sample well formed at each pixel of the plurality ofpixels; and a sensor formed at each pixel of the plurality of pixels,wherein the sensor is arranged to receive a first emission from thesample well, and wherein the sensor is further configured to acquire afirst signal and a second signal during a charge-accumulation cycle ofthe sensor for the first emission and to output the first signal and thesecond signal as representative of the received first emission.
 2. Theintegrated device of claim 1, further comprising integrated circuitryformed on the substrate with the sensor and arranged to receive thefirst signal and the second signal from the sensor.
 3. The integrateddevice of claim 2, wherein the integrated circuitry includes acommunication interface for transmitting data to a computing deviceexternal to the integrated device.
 4. The integrated device of claim 1,further comprising an excitation source arranged to provide excitationenergy to the sample well that excites the first emission.
 5. Theintegrated device of claim 4, wherein the excitation source providesoptical radiation having a characteristic wavelength betweenapproximately 350 nm and approximately 1000 nm.
 6. The integrated deviceof claim 4, wherein the excitation source is located between the samplewell and the sensor.
 7. The integrated device of claim 1, wherein thesample well is adapted to retain nucleotides or nucleotide analogs. 8.The integrated device of claim 1, wherein the sensor comprises at leasttwo spatially separated photodetector segments, and the first signal andthe second signal are representative of a spatial distribution patternfor a first spectral band of the first emission.
 9. The integrateddevice of claim 1, wherein each pixel of the plurality of pixelscomprises a sample well and a sensor arranged to receive opticalemission from the sample well.
 10. The integrated device of claim 1,further comprising a micro-optical or nano-optical structure thatincreases an intensity of excitation energy provided to the sample well.11. The integrated device of claim 1, further comprising a walledchamber formed around the plurality of pixels that is configured to holda specimen.
 12. The integrated device of claim 11, wherein theintegrated device and walled chamber are packaged in a single modulehaving exterior electrical contacts that are arranged for electricalconnection with contacts of a receiving dock of an instrument.
 13. Theintegrated device of claim 1, further comprising a divot extending fromthe sample well into an optically-transparent layer of material.
 14. Theintegrated device of claim 1, wherein the integrated device and samplewell are arranged to receive samples in a fluid suspension.
 15. Theintegrated device of claim 1, wherein a minimum diameter of the samplewell is between approximately 30 nanometers and approximately 250nanometers.
 16. The integrated device of claim 1, further comprisingread-out circuitry formed on the substrate and arranged to read outsignals from sensors in each of the plurality of pixels.
 17. Theintegrated device of claim 16, wherein the sensor and read-out circuitrycomprise CMOS circuit elements.
 18. A method of analyzing a plurality ofsamples in parallel, the method comprising: receiving, at a first sensorformed at a first pixel of a plurality of pixels formed on a substrateof an integrated device, a first emission from a first sample wellformed at the first pixel; producing, by the first sensor, a firstsignal and a second signal representative of the received firstemission; acquiring the first signal and the second signal during acharge-accumulation cycle of the first sensor for the first emission;and transmitting the first signal and the second signal from the firstsensor for analysis.
 19. The method of claim 18, further comprisingproviding excitation energy to the sample well to excite the firstemission.
 20. The method of claim 18, further comprising evaluating aratio of the first signal and the second signal to determine a propertyof a sample in the sample well.
 21. The method of claim 18, furthercomprising performing an extension reaction at a priming location of atarget nucleic acid molecule in the sample well and in the presence of apolymerizing enzyme to sequentially incorporate nucleotides ornucleotide analogs into a growing strand that is complementary to thetarget nucleic acid molecule.
 22. The method of claim 21, furthercomprising: receiving signal sets from the sensor following excitationof the sample well; and identifying the types of nucleotides ornucleotide analogs based on said received signal sets, therebysequencing said target nucleic acid molecule.